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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Microtubule-based positioning mechanisms
    Teapal, J. - \ 2014
    Wageningen University. Promotor(en): Marcel Janson; Bela Mulder. - Wageningen : Wageningen University - ISBN 9789462570474 - 135
    microtubuli - celbiologie - cytologie - cellen - eiwitten - celskelet - celkernen - organellen - microtubules - cellular biology - cytology - cells - proteins - cytoskeleton - nuclei - organelles

    A brief look at the animal and plant kingdom shows a large variety of spatial patterns like the periodic stripes of zebras and the arrangement of flower petals. Interestingly, a microscopic look at tissues and single cells reveals very well structured organisations at smaller length scales as well. In this thesis I provide mechanistic insights into the organization of such patterns.

    To build complex structures, cells require mechanism to set a length scale. Apart from mechanisms based on the reaction and diffusion of interacting molecules, mechanical processes like the growth of cytoskeletal filaments can set length scales at the subcellular level. The cytoskeleton mechanically supports cells but more importantly for our purpose generates forces that can change cellular architecture. In eukaryotic cells the contribution is based on three filaments (microtubules, actin and intermediate filaments), but in this thesis the focus is set on microtubules. Microtubules are stiff and dynamic filaments that enable them to play a prominent role in cellular organization.

    Microtubules are composed of tubulin heterodimers that arrange longitudinally and laterally to form a slender hollow tube with high rigidity. Microtubules in cells grow away from specialized nucleation sites and have a certain probability to undergo a transition to a state of shortening. This switching mechanism, termed dynamic instability, determines how far microtubules grow away from their nucleation site. This length regulating mechanism aids the positioning of cellular components in cooperation with molecular motors that transport material along microtubules and forces generated by growing and shrinking microtubules in contact with cellular objects (organelles, membrane). The role of microtubules in intercellular positioning mechanisms is reviewed in chapter 1.

    In chapter 2 and 3 we investigated the role of microtubules in positioning nuclei in cells. compartments throughout the cellular space. The spacing between compartments is in many cases Eukaryotic but also prokaryotic cells disperse organelles and micro- regulated and equidistant patterns have been described in particular for the case of nuclei in multinucleated cells. The spacing between nuclei is regulated to control the patterning of cells in developing embryos but the occurrence of irregular patterns in large multinucleated muscle cells also correlates with muscle diseases. In chapter 2 we used fission yeast cells with a cytokinetic defect to generate a model multinucleated cell. Fission yeast cells are easy to genetically modify and the organization of their microtubule network is well understood. Cells had a cluster of nuclei at their centre but in absence of the minus-end directed motor klp2p the pattern changed to an arrangement in which the nuclei were well dispersed and positioned at equidistant intervals. Patterning depended on the presence of microtubules and we observed the growth of microtubules away from the envelope of nuclei towards neighbouring nuclei. We hypothesized that impingement of microtubules onto neighbouring nuclei generates nuclear repulsive forces. The net effect of microtubule interactions with cell walls and nuclei may be a force field in which nuclei are stably positioned at equidistant positions. However dominant forces generated by klp2p cause sliding between microtubules originating from sister nuclei that pull nuclei together. Our studies thus suggest a mechanism for equidistant positioning of organelles and a way to switch between patterns. Switching behaviour is observed in biology for example during light induced redistribution of chloroplasts in plant cells.

    An increasing number of biological findings are now supported by computer models, as it allows to deduce whether a limited set of interacting components can explain a biological phenomenon. To evaluate whether repulsive pushing forces by dynamic unstable microtubules in between nuclei are sufficient to pattern nuclei we developed a simple 1D stochastic model of microtubule growth and nuclear motion in a tetranucleated cell. Our model demonstrated that the dynamics and accuracy of nuclear positioning in fission yeast cells is in agreement with the measured parameters of dynamic instability of microtubules. For this we compared nuclear oscillations and nuclear redistribution after pattern perturbation in experiments and simulations. An overestimation of the force generation between nuclei in our model caused a larger internuclear distance then experimentally observed. This discrepancy disappeared when we took into account that force generation at cell walls is more efficient than at nuclear envelopes. The model in chapter 3 thus clearly revealed that equidistant nuclear positioning can be explained by force generation of microtubules undergoing dynamic instability.

    In chapter 4 we investigated the role of microtubule dynamics in the regulation of spindle elongation. During mitosis, microtubules form the mitotic spindle that segregates chromosomes to different cell halves. Microtubules from two spindle halves interdigitate at the spindle centre and template a multi protein assembly called the spindle midzone. Microtubules within the midzone grow and slide relative to each other causing spindle elongation. Moreover, the midzone regulates cytokinesis and as such mechanism that control midzone assembly are of interest to identify new targets for cancer therapy. In chapter 4 we investigated, using fission yeast as a model, the mechanism that regulates the length of the midzone during spindle elongation. We demonstrate that spindle elongation velocity is limited by the speed at which motors push overlapping microtubules apart. However, under conditions of reduced microtubule growth, the elongation is being limited by microtubule growth. These results show that sliding and microtubule growth are coupled to prevent that spindle halves can separate from each other by sliding alone. This insight will help to reveal the function of a myriad of protein interactions that take place at the midzone.

    This thesis reveals mechanically insights into pattern formation based on microtubule dynamics. In chapter 5 we discuss the relevance of our findings for the patterning of nuclei in larger eukaryotic cells. Preliminary results on the binding of the major midzone protein ase1p show that the binding affinity of ase1p microtubule crosslinkers depends on the number of microtubules that it can potentially bind to. These results suggest how ase1p may be recruited to the centre of mitotic spindles, where microtubule interdigitation is strongest.

    Changes in Nuclear Structure During Wheat Endosperm Development
    Wegel, E. - \ 2005
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Hans de Jong; P.J. Shaw. - - 75
    triticum aestivum - endosperm - tarwe - celkernen - celstructuur - plantenontwikkeling - cytologie - modellen - chromatine - cytogenetica - dna - triticum aestivum - endosperm - wheat - nuclei - cell structure - plant development - cytology - models - chromatin - cytogenetics - dna
    This thesis is an investigation into the structure of wheat endosperm nuclei starting with nuclear divisions and migration during syncytium formation followed by the development of nuclear shape and positioning of chromosome territories and ending with changes in subchromosomal structure during the activation of a transgene locus.

    At the level of the whole endosperm we have developed a method for modelling in 3D the formation of the syncytium that characterises early endosperm development. After the initial nuclear division of the first endosperm nucleus three groups of nuclei form in the original central cell: a stem-like group of nuclei close to the zygote and connected to a single layer of nuclei in the dorsal periphery and another, unconnected single layer in the ventral periphery. By two days post anthesis (dpa) both the ventral and dorsal groups of nuclei have developed into plates of nuclei. The dorsal plate then merges with the ventral plate through synchronous nuclear divisions. By 4 dpa the entire periphery of the central cell is surrounded by a layer of nuclei and the syncytium is complete.

    Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA-content to 6C and 12C. 3D modelling of nuclei with DNA contents of 3C, 6C and 12C allowed us to visualise progressive changes in nuclear shape and chromosome positioning. With increasing C content, nuclear volumes increase predominantly in two directions, thereby changing the shape of the nuclei into a disc-like structure. Wheat chromosomes in interphase nuclei have a typical Rabl configuration with centromeres and telomeres arranged at opposite poles. The majority of centromeres and telomeres are found at or close to the nuclear membrane, some also in the middle of the nucleus and in rare instances a telomere can be observed at the centromere pole and vice versa. This means that centromeres and telomeres are not or only transiently anchored to the nuclear membrane. Both centromeres and telomeres show a degree of non-homologous associations, which for centromeres remains constant through increases in ploidy, while telomere associations increase with higher C-values.

    The wheat line we used has a 1RS / 1BS ( Secale cereale ) chromosome arm substitution. Fluorescence in situ hybridisation detection of the rye arm substitution with total genomic DNA as probe revealed the following: during endosperm development chromosomes are able to change their position in all three dimensions and as the C-content increases in nuclei that have stopped dividing chromosome arrangements become progressively distorted. The vast majority of 12 C nuclei show six rye chromosome arms, but a few show three groups of associated rye chromosome arms. This means that during endosperm development wheat nuclei increase their ploidy through both polyploidisation and polytenisation.

    The final part analyses the chromatin structure of active and inactive gene loci. The High Molecular Weight (HMW) glutenin genes in wheat are developmentally activated in the endosperm at about 8 dpa. I have investigated the physical changes that occur in these genes in two transgenic lines containing about 20 and 50 copies respectively of the HMW glutenin genes together with their promoters. Fluorescence in situ hybridisation (FISH) and confocal imaging data show that in non-expressing tissue each transgene locus consists of one or two highly condensed sites that decondense into many foci upon activation of transcription in endosperm nuclei. Initiation of transcription can precede decondensation but not vice versa. In one of the lines, cytoplasmic transcript levels are high after onset of transcription, but disappear by 14 dpa, while siRNAs, indicative of post-transcriptional gene silencing (PTGS), are detected at this stage. However, the transcript levels remain high at the transcription sites, the great majority of the transgene copies are transcriptionally active and transcriptional activity in the nucleus ceases only with cell death at the end of endosperm development.
    Het transportoom
    Emons, A.M.C. - \ 2002
    Wageningen : Wageningen Universiteit - 30
    cellen - transport - omloop - celstructuur - cytologie - celbiologie - cells - transport - circulation - cell structure - cytology - cellular biology
    Probing nod factor perception in legumes by fluorescence microspectroscopy
    Goedhart, J. - \ 2001
    Wageningen University. Promotor(en): A.H.J. Bisseling; T.W.J. Gadella Jr.. - S.l. : S.n. - ISBN 9789058084811 - 142
    peulgewassen - rhizobium - stikstof - groei - symbiose - wortels - wortelharen - wortelknolletjes - membranen - n-acetylglucosamine - cytologie - fluorescentiemicroscopie - moleculaire biologie - legumes - rhizobium - nitrogen - growth - symbiosis - roots - root hairs - root nodules - membranes - n-acetylglucosamine - cytology - fluorescence microscopy - molecular biology

    Plants of the family of legumes are capable of forming a symbiosis with Rhizobium bacteria. These Gram-negative bacteria invade the root system of a host legume and fix nitrogen in a specialized organ, the so-called root nodule. In exchange for sugars, the bacteria convert atmospheric nitrogen to ammonia which can be used by the plant. This remarkable alliance allows the plant to grow independently from nitrogen sources provided by the soil. Examples of leguminous plants are clover, pea, and soybean.

    The symbiosis is initiated by a molecular dialogue. The plant produces flavonoid compounds which are recognized by the bacterial NodD protein. The signaling pathway which is activated leads to the synthesis and secretion of lipo-chitooligosaccharides which are also called Nod factors. The production of Nod factors by the Rhizobium bacteria is an essential step for accomplishing symbiosis and also determines host specificity. The general structure of Nod factors comprises a chitin backbone of three to five b-1,4-linked N-acetylglucosamine units. A fatty acid of 16-20 carbon atoms is N-linked to the terminal non-reducing sugar residue. The exact molecular structure can comprise different acyl chains and a variety of decorations on the chitin backbone depending on the Rhizobium species.

    After successful recognition of the bacteria by the legume, a remarkable morphogenic process takes place, which is known as root hair curling. The root hair curls around the Rhizobium colony by which the bacteria are entrapped within the so-called shepherd's crook. Subsequently, the rhizobia enter the root hair through an infection thread, starting from the center of the curl. Via the infection thread several cell layers are crossed after which the bacteria are released in nodule primordium cells, where they differentiate into bacteroids that fix nitrogen.

    Nod factors in the absence of bacteria, either purified from Rhizobium cultures or chemically synthesized can elicit a wide variety of responses on a compatible legume host. When Nod factors are applied to roots, the earliest visible response takes place in root hairs. Root hairs are single tip-growing cells that develop from the epidermis of a root and grow perpendicular from the longitudinal axis of the root. Generally, root hairs that are terminating growth are susceptible to Nod factors and respond by swelling of the tip of the root hairs, followed by the re-initiation of tip growth in a random direction. This typical Nod factor response is referred to as root hair deformation and can be observed with a microscope 2-3 hours after addition of Nod factors.

    The perception of Nod factors by the plant, and the downstream signaling cascades that are activated are major research topics in the Rhizobium-legume interaction. The low concentration (down to 10-12 M) at which Nod factors can still induce root hair deformation and the dependence of the bioactivity on specific decorations of the Nod factor suggest that these molecules are perceived by receptors at the root hair. However, to date no such receptors are characterized. Moreover, it is far from clear where Nod factor recognition by root hairs takes place. Therefore an approach was taken in which fluorescent Nod factor derivatives are synthesized, allowing to probe the ligand binding sites on legume root hairs.

    The research described in this thesis focuses on the quantification, characterization and perception by legumes of Nod factors. In order to detect Nod factors at physiologically relevant concentrations sensitive techniques are required. A number of fluorescence spectroscopy and microscopy based techniques can be used to study fluorescent derivatives of signaling molecules. In chapter 1, the use of fluorescence microspectroscopic techniques available in the laboratory are discussed. Examples how these techniques can be used for the study of root hairs and other living cells are described.

    In chapter 2, two methods to quantify purified Nod factors are described. An enzymatic step which is crucial for the first method was analyzed in detail. The second method was optimized and validated using fluorescent and radiolabeled Nod factor derivatives. The chapter describes in detail how the two optimized methods can be used for quantifying Nod factors as well as potential pitfalls.

    In chapter 3, the spectral properties of three novel fluorescent Nod factor derivatives are described. It is checked whether these fluorescent Nod factors can still elicit root hair deformation on Vicia sativa roots. The properties of the amphiphilic signaling molecules were characterized in vitro in the absence and presence of micelles and model membrane systems using fluorescence spectroscopy. Time-correlated single photon counting fluorescence spectroscopy was used to measure rotational mobility of the fluorophore. These experiments are complemented with fluorescence correlation spectroscopy to examine diffusional mobility of the Nod factors. A lipid transfer assay was used to measure the rate of intermembrane transfer and intramembrane flip-flop of Nod factors.

    In chapter 4, a detailed study is reported describing the sites at which the fluorescent Nod factors accumulate. Fluorescence microscopy is used to examine the location of fluorescent Nod factors on root hairs during the initial perception and during root hair deformation. Subsequently, the diffusional mobility of the fluorescent Nod factors is measured in vivo using fluorescence correlation microscopy (FCM), allowing quantification of molecular mobility and concentration of fluorescent Nod factors in living root hairs at a molecular level. This study is continued in chapter 5 in which also novel sulfated fluorescent Nod factors are used and characterized, enabling a direct comparison between sulfated and non-sulfated Nod factors on a host and non-host legume. Also, the origin of the molecular mobility of the Nod factors is studied in more detail.

    In chapter 6 a novel approach towards manipulating phospholipid second messengers in single cells with spatiotemporal control is presented. The synthesis of a fluorescent and caged derivative, NPE-phosphatidic acid, which releases phosphatidic acid upon exposure to UV is described. The release of phosphatidic acid from the caged compound is studied in vitro and in vivo. The use of photoreleasable phosphatidic acid for studying phospholipid signaling in vivo is evaluated.

    Chapter 7 summarizes the conclusions that can be drawn from the results described in this thesis. The implications for Nod factor secretion by the bacterium and subsequent perception by legume root hairs are discussed. Based on the results presented in this thesis, it is tempting to speculate that spatial restriction of signaling molecules in plants is achieved by immobilization in the cell wall. Subseqent perception of Nod factor takes place either in the plasma membrane or within the cell wall as is illustrated by two proposed modes of perception. The results of this thesis are discussed with respect to these two models.

    Meclatis in Clematis: yellow flowering Clematis species : systematic studies in Clematis L. (Ranunculaceae), inclusive of cultonomic aspects
    Brandenburg, W.A. - \ 2000
    Agricultural University. Promotor(en): L.J.G. van der Maesen. - S.l. : S.n. - ISBN 9789058082374 - 287
    clematis - taxonomie - fylogenetica - plantengeografie - morfologie - cytologie - palynologie - clematis - taxonomy - phylogenetics - phytogeography - morphology - cytology - palynology

    The general classification of the genus Clematis (Ranunculaceae) was subject of study in chapter 1. Based on species character scores, the infrageneric classification was analyzed by applying Hennig86 as phylogenetical analysis package. As result of this analysis Clematis was subdivided into 18 sections, one of them subdivided in 3 subsections.

    The world distribution of Clematis was also studied with Hennig86. It was not possible to postulate the area of origin of the genus Clematis with the available data set.

    A interspecific cross polygon was made and analyzed by seed set and pollen tube growth. Its systematic significance with regard to Clematis and in general was discussed. Dependent on the adopted species concept, these crosses are crucial or just academic. The adoption of the phylogenetic species concept made that this choice for Clematis is academic. Nevertheless, it is useful information for plant breeders.

    A general description of the genus Clematis was presented with some background information on certain characters, such as overall habitus, nectar leaves and the position of nectaries.

    Chapter 2 was devoted to Clematis sect. Meclatis . This particular section consists of the yellow-flowering Clematis spp., that are gaining popularity in gardening. Many efforts were directed to reveal the species delimitation. It appeared that the phenetic methodology is of restricted value in such a complex of quite similar species. Using a combination of methods, the phylogenetic analysis by Hennig86 finally revealed the species delimitation: Clematis orientalis , C. graveolens , C. intricata , C. ispahanica , and C. tibetana . C. tibetana was subdivided into three subspecies: subsp. tibetana , subsp. tangutica and subsp. vernayi . Well-known 'horticultural species' such as C. tangutica and C. vernayi were reduced in rank and others such as C. glauca and C. akebioides were reduced to synonymy. A summary of chromosome, pollen and isozyme data was presented.

    Chapter 3 was focusing on more fundamental aspects of systematics of cultivated plants. It has been shown that the cultivar group is of crucial importance in classifying cultivars, that the classification principle for cultivated plants is open instead of closed and consequently that the basal term in systematics of cultivated plants for an entity cannot be taxon, but should be a new term culton (plur. culta; cultonomy for culta vs. taxonomy for taxa).

    Clematis is one of the first genera for which a cultivar group classification was presented in a systematic way, as was outlined in chapter 4. A short survey was given of the introduction into cultivation of yellow-flowering Clematis spp., and a major part of the yellow-flowering Clematis cultivar assortment has been described. So far no cultivar groups are needed for these cultivars.

    Aspects of plant cell growth and the actin cytoskeleton : lessons from root hairs
    Ruijter, N.C.A. de - \ 1999
    Agricultural University. Promotor(en): M.T.M. Willemse; A.M.C. Emons; J.H.N. Schel. - S.l. : S.n. - ISBN 9789058080981 - 164
    planten - cytologie - wortelharen - celgroei - celskelet - actine - rhizobium - vicia sativa - plantaardig eiwit - onderzoek - plants - cytology - root hairs - cell growth - cytoskeleton - actin - rhizobium - vicia sativa - plant protein - research

    The main topic the thesis addresses is the role of the actin cytoskeleton in the growth process of plant cells. Plant growth implies a combination of cell division and cell expansion. The cytoskeleton, which exists of microtubules and actin filaments, plays a major role in both processes. Before cell growth takes place, a new cell is formed by cell division. The orientation of the division plane most often predicts the orientation of cell expansion, and a correct positioning of the division plane is therefore important for plant morphogenesis. During most stages of cell division microtubules and actin filaments have a similar configuration.

    In Chapter 1 (De Ruijter et al. , 1997, Acta Bot. Neerl . 46: 279-290) the cytoskeleton of microtubules has been visualized during all stages of cell division for long and short root tip cells of broad bean ( Vicia faba L.). In all cells the preprophase band of microtubules was positioned in the midplane of the cell, and perpendicular to the long axis of the root. It was observed that the spindle axis in short cells increasingly tilted, from meta- to anaphase, giving rise to oblique cell plates . It appears that this is caused by spatial constraints. During late-telophase, cell plates first rotated towards the transversal plane before they fused with the parental wall at the site of the earlier preprophase band. When cell division is completed, cells grow.

    Plant cell growth is the insertion of Golgi vesicles into the plasma membrane and the delivery of their content into the existing wall. If this wall is flexible and under turgor pressure, the membrane becomes larger and the wall expands. The basic principles of plant cell growth can best be studied in cells where this growth process takes place abundantly, that is in the tip of tip-growing cells of higher plants, such as root hairs and pollen tubes. In Chapter 2 (De Ruijter et al ., 1998, Plant J. 13: 341-350) characteristics for cell tip growth are being reported, studied by comparison of developmental stages of root hairs of vetch ( Vicia sativa L .), from their emergence to their maturity. It is further shown that lipochito-oligosaccharides (LCOs), well-characterized molecules that are excreted by bacteria, reinitiate cell tip growth in hairs that are terminating growth. Tip growth and the site of growth re-initiation correlates with the presence of a steep cytoplasmic calcium gradient at the plasma membrane. Furthermore, it was found that a spectrin-like protein is a good marker for tip growth, and co-localizes with the vesicle rich region, which is known to be present at the tip.

    Immunolocalization of this spectrin-like protein in plants was extended to a variety of growing cells and shows, in Chapter 3 (De Ruijter and Emons, 1993, Cell Biol. Int. 17: 169-182), that this protein is especially present in young growing cells. Molecular weight and iso-electric point determination, by means of immuno-blotting identified the plant spectrin-like protein. The anti-spectrin antibody also labels nuclei, which is further investigated in Chapter 4.

    To analyze the presence and localization of nuclear spectrin-like proteins, various plant tissues and isolated pea nuclei were labeled. The data presented in Chapter 4, show that the spectrin-like proteins are distributed in a speckled pattern and occasionally in tracks. The extraction procedures used indicate that the spectrin-like protein is part of the nuclear matrix in which it may be a stabilizing factor.

    In Chapter 5 (Miller, De Ruijter et al., 1999, Plant J. 17: 141-154) the actin cytoskeleton of vetch root hairs at their initiation and during their development is described. Actin filament bundles are the dynamic backbone of the cytoplasmic strands. Growing hairs show dense sub-apical fine bundles of actin filaments (FB-actin) and the very tip is devoid of actin filament bundles, whereas full-grown hairs have actin bundles looping through the tip. Similar actin configurations were obtained when root hairs were freeze substituted and immunolabeled with anti-actin, or chemically fixed by an improved method and stained with fluorescent phalloidin. Since LCOs had been shown to reinitiate root hair growth (Chapter 2), this signal molecule was used to study the actin cytoskeleton during growth reinitiation. Manipulation of the actin cytoskeleton with the actin filament capping drug cytochalasin D inhibited polar growth. However, root hair initiation and swelling after LCO application were not affected. We concluded that elongating FB-actin is another characteristic for tip growth.

    Indeed, LCOs altered the configuration of the actin cytoskeleton, which was studied in Chapter 6 (De Ruijter et al. , 1999, MPMI 12: 829-832). The density of sub-apical actin filament bundles increased within 3-15 minutes after the application of LCOs. By a quantitative approach we were able to define the minimal FB-actin density and minimal length, of the area with the FB-actin, needed for growth. Only in hairs in which FB-actin exceeded these values, tip growth was sustained or resumed. The rapid response of actin filaments indicates a role for the actin cytoskeleton in signal transduction cascades.

    Such a dynamic actin cytoskeleton must be regulated. Part of this regulation will be done by actin binding proteins. Therefore, our limited knowledge of actin binding proteins in plant cells is reviewed in Chapter 7 (De Ruijter and Emons, 1999, Plant Biology 1: 26-35) of the thesis.

    Chapter 8 summarizes the characteristics for growth in tip-growing cells and extrapolates them to cells that expand isodiametrically or predominantly along one length axis. We conclude that tip growing cells, like root hairs, shed light on basic principles of plant growth, and provide a system to monitor the effect of signal molecules on cell growth.

    Interaction between root-knot nematodes and Solanum spp. : variation in pathogenicity, cytology, proteins and DNA = [De interactie tussen wortelknobbelnematoden en Solanum spp. : variatie in ziekteverwekkend vermogen, cytologie, eiwitten en DNA]
    Beek, J.G. van der - \ 1997
    Agricultural University. Promotor(en): R.F. Hoekstra; C.H. van Silfhout; P.W.T. Maas. - S.l. : Van der Beek - ISBN 9789054857280 - 219
    wortelknolletjes - knobbelvorming - plantenparasitaire nematoden - meloidogyne - plantenplagen - solanaceae - pathogeniteit - genetische variatie - cytologie - eiwitten - dna - plantennematologie - gastheer parasiet relaties - dissertaties - root nodules - nodulation - plant parasitic nematodes - meloidogyne - plant pests - solanaceae - pathogenicity - genetic variation - cytology - proteins - dna - plant nematology - host parasite relationships - theses

    This thesis describes genetic variation in the root-knot nematodes Meloidogyne hapla, M. chitwoodi and M. fallax, particularly with respect to their pathogenicity on Solanum spp. Significant differences in virulence and aggressiveness were shown to exist between and within these species. Evidence for the occurrence of pathotypes of M. chitwoodi on S. bulbocastanum was obtained. Differences in virulence corresponded to differences in overall genetic variation, revealed by 2-D protein electrophoresis. A distinct species classification for Meloidogyne spp. was obtained by AFLPs and 2-D electrophoresis. In mating experiments M.chitwoodi and M. fallax appeared to be true biological species as testified by infertility of their hybrids. Abnormalities during meiosis in oocytes of an isolate of M. hapla and in spermatocytes of isolates of M. fallax resulted in limited sexual recombination. The combination of post-reductional meiosis and the fusion of the second polar body with the egg pronucleus is probably responsable for maintenance of heterozygosity in meiotic parthenogenetic Meloidogyne. The constant production of males in these populations makes the development of homogeneous isolates impossible. A method was described to conserve nematode germplasm by long-term preservation of juveniles in liquid nitrogen.
    Non-homologous chromosome synapsis during mouse meiosis : consequences for male fertility and survival of progeny
    Peters, A.H.F.M. - \ 1997
    Agricultural University. Promotor(en): C. Heyting; P. de Boer. - S.l. : Peters - ISBN 9789054857761 - 182
    muridae - muizen - meiose - geslachtelijke voortplanting - parthenogenese - polyembryologie - vruchtbaarheid - overleving - levensvatbaarheid - interacties - milieu - uitsterven - stofverplaatsing - chromosoomtranslocatie - chromosomen - cytologie - histologie - muridae - mice - meiosis - sexual reproduction - parthenogenesis - polyembryony - fertility - survival - viability - interactions - environment - extinction - translocation - chromosome translocation - chromosomes - cytology - histology
    In the mouse, heterozygosity for several reciprocal and Robertsonian translocations is associated with impairment of chromosome synapsis and suppression of crossover formation in segments near the points of exchange during prophase of meiosis. This thesis describes the analysis of the consequences of the occurrence of non-homologous synapsis and/or suppression of meiotic crossover formation over many successive generations for male fertility and viability of the progeny.

    For studying chromosome synapsis, we modified a drying down technique which results in high yields of nuclei of all first meiotic prophase stages in both male and female from only small amounts of tissue (chapter 2). Preparations are suitable for synaptonemal complex (SC) analysis by normal light and electron microscopy (chapters 2, 3 and 7), for fluorescence immunocytochemistry and in situ hybridization (chapters 2, 8).

    In the study presented in chapter 3, we analysed the variation in male fertility of mice double heterozygous for two near identical reciprocal translocations T(1;13)70H and T(1;13)1Wa in relation to the synaptic behaviour of two differently sized heteromorphic bivalents during meiotic prophase. Male fertility rises when non-homologous synapsis in the small 1 13heteromorphic bivalent, leading to a "symmetrical" SC, is more frequent at the initial prophase stages. Based on the data presented, we favour the "unsaturated pairing site" model as the primary cause for male sterility.

    In T70H/T1Wa females not all heterologous synapsis within the small heteromorphic bivalent is effectuated during the early stages of meiosis; some is achieved lateron by the mechanism of "synaptic adjustment" (chapter 3). Each heteromorphic bivalent contains a copy of the chromosome 1 region between the T70H and T1Wa breakpoints which is about 10 cM in size (Δ1 segment). Although axial elements representing these Δ1 segments are seen to approach each other during early meiotic prophase stages, they never successfully constitute a synaptonemal complex in either sex (chapter 3). This agrees with the fact that in earlier cytogenetic studies quadrivalents were never seen at both male and female diakinesismetaphase 1.

    In chapter 7, we demonstrate that male fertility of the T70H/T1Wa mice is not only determined by the chromosomal constitution of the carrier but is additionally influenced by the pairing or synaptic history in previous meioses of especially the T70H and T1Wa short translocation chromosomes. Fertility of T70H/T1Wa males is more impaired after one or more successive transmissions of the T1Wa translocation chromosomes through a heteromorphic bivalent configuration, irrespective of the sex of the transmitting parent.

    Furthermore, we show that the introduction of the Robertsonian translocation Rb(l1.13)4Bnr into the T70H/T1Wa karyotype restores fertility of double heterozygous males by stimulating non-homologous synapsis of the small heteromorphic bivalent. We speculate that this Rb4Bnr effect is mediated by a prolongation of the early stages of meiotic prophase I.

    Successive female transmissions of the T1Wa translocation chromosomes in the presence of Rb4Bnr inititially resulted in an increase of the capacity for early meiotic nonhomologous synapsis within the small heteromorphic bivalent, leading to a restoration of fertility for the majority of carriers. Subsequently, a decrease of the capacity of the small heteromorphic bivalent to fully synapse was noticed, although a higher than original (F1) background level of male fertility remained.

    These variations in male fertility are most likely based on epigenetic variance, reflected as the capacity to engage into non-homologous synapsis early in male meiosis leading to a "symmetrical" SC, despite the different amounts of chromatin to accommodate.

    In chapter 4, the localization of several microsatellite markers and single copy genes relative to the T70H and T1Wa breakpoints, using quantitative PCR, quantitative Southern blotting and in situ hybridization, is described.

    In chapter 5, we investigated the level of suppression of meiotic recombination and impairment of chromosome synapsis in T70H heterozygotes in relation to the viability of the progeny. For T70H/+ females, the introgression of the D1Mit4, D1Mit20 and D1Mit122 microsatellite marker alleles positioned distal of the T70H breakpoint on the normal chromosome 1 into the 13 1T70H long translocation chromosome was suppressed in a distance dependent manner. This effect was more pronounced in T70H/+ females, additionally homozygous for Rb4Bnr. The delay in introgression was paralleled by a reduction of the frequency and extent of non-homologous synapsis in segments near the T70H breakpoints of the pachytene translocation multivalents in T70H/+ and Rb4BnT70H/Rb4Bnr+ males. The extend of non-homologous synapsis around the centre of the synaptic cross configuration in these males correlated with fluctuations in prenatal viability of segregating translocation homozygotes in crosses between (Rb4Bnr)T70H homozygous males and heterozygous females when meiotic drive at the female second meiotic division is excluded. The reduction in viability is explained by the gain of mutations resulting from incorrect processing of recombination intermediates which is due to non-homologous synapsis around the translocation breakpoints.

    In chapter 6, we analysed the consequences of the absence of crossing over for regions between the T70H and T1Wa breakpoints (Δ1 and Δ13 segments) of the Rb4BnrT1Wa translocation chromosomes, which have been transmitted for over 20 generations via heteromorphic bivalents in Rb4BnrT70H/Rb4BnrT1Wa females. Survival of heterozygous and homozygous carriers for these segments was taken as the phenotypic endpoint. The viability of progeny of crosses between Rb4BnrT70H homozygous males and Rb4BnrT70H/Rb4BnrT1Wa females, of which the latter principally produce 4 types of gametes, was estimated using a haplotype analysis of microsatellites in the Δ1 segment for genotyping (see chapter 4). We observed no differences in the pre- and postnatal survival rates of the double heterozygous and 13 1H, 13 1H, 1 13Wa 1 13H "duplication" progeny in which the Δ1 and Δ13 segments of the T1Wa translocation chromosomes had either no, an onegeneration or a multi-generation history of non-homologous synapsis in heteromorphic bivalents during previous female meioses. In addition, intercrossing of Rb4BnrT70H/Rb4BnrT1Wa double heterozygotes after genetic isolation of these Δ1 and Δ13 segments for 20 to 22 generations, showed that the viability of the Rb4BnrT1Wa homozygotes was not different from the Rb4BnrT70H homozygous and Rb4BnrT70H/Rb4BnrT1Wa karyotypes generated by this cross. Thus, exclusion of the Δ1 and Δ13 segments from meiotic crossing over within non-homologous synapsed heteromorphic bivalents during 20 to 25 successive generations does not result in an accumulation of recessive lethal mutations or an increased susceptibility for gaining dominant lethal mutations.

    For the D1Mit122 microsatellite used in offspring haplotyping a higher mutation frequency was observed after transmission through a double heterozygous than after transmission through a T70H homozygous karyotype (chapter 6). On the basis of the identity of the mutations, the ectopic pairing of the St2 gene copies (containing D1Mit122) during meiosis of T70H/T1Wa males (chapter 8) and the observation of ectopic homologous contacts of the Δ1 segments during the zygotene stage without SC formation (chapter 3), we speculate that these mutations are the result of ectopic homologous gene conversion events most likely occurring in the absence of a synaptonemal complex.

    The crossover suppressive influence of the Rb translocation on the Δ1 segment (chapter 5) enabled us to analyze the effects of introgression of genetic material from the Swiss +/+ stock into the translocation karyotypes. Introgression of "new" genetic material correlated with an increase in littersize of Rb4BnrT70H homozygotes (chapter 5), an improvement of the life expectancy of Δ1 duplication offspring from double heterozygous mothers (chapter 6) and a clear improvement of male fertility in double heterozygous and T70H homozygous males also carrying Rb4Bnr (chapter 7). These pleiotrophic findings are discussed in chapter 8 in terms of genetic versus epigenetic mechanisms of inheritance.

    Finally, when T1Wa was backcrossed for many generations to the Rb4BnrT70H/Rb4BnrT70H karyotype, essentially precluding genetic recombination in the Δ1 and Δ13 segments, or when T1Wa was combined with Rb4Bnr after many successive transmissions via alternating T1Wa heterozygotes and homozygotes, stable Rb4BnrT1Wa homozygous lines could not be bred (chapter 8). Especially female reproductive performance decreases after repeated male and female homologous meiosis. As non-homologous synapsis in the centre of the synaptic cross configuration in T1Wa/+ males is common too (unpublished results), more work into the genetic stability of chromosome segments, that have a history of hindered homologous interaction, is indicated (chapter 8).

    Seed development in Phaseolus vulgaris L., Populus nigra L., and Ranunculus sceleratus L. with special reference to the microtubular cytoskeleton
    XuHan, X. - \ 1995
    Agricultural University. Promotor(en): M.T.M. Willemse, co-promotor(en): A. Souvre; A.A.M. van Lammeren. - S.l. : XuHan - ISBN 9789054854036 - 150
    plantenfysiologie - plantenontwikkeling - bloemen - vruchten - plantenorganen - cytologie - celmembranen - phaseolus vulgaris - bosbouw - bomen - celfysiologie - bloeiende planten - plant physiology - plant development - flowers - fruits - plant organs - cytology - cell membranes - phaseolus vulgaris - forestry - trees - cell physiology - flowering plants

    In this thesis, seed development is investigated in celery-leafed buttercup ( Ranunculus sceleratus L.), bean ( Phaseolus vulgaris L.) and poplar ( Populus nigra L.). Developing embryos, endosperms and seed coats are investigated. The comparative study of seed morphology, anatomy and development gives insight into the different types of seed differentiation in relation to its function. A main goal of the thesis is to study the role of the microtubular cytoskeleton in plant reproduction processes. Special attention has been paid to configurations of microtubular cytoskeleton during cellularization of the endosperm. Functions of the microtubular cytoskeleton in relation to the particular organizations of microtubular populations have been analyzed based on the studies of the overall developmental patterning of tissues and organs. Cytomorphogenesis during seed development is investigated at histological and cytological levels using combined techniques of conventional light microscopy, scanning and transmission electron microscopy, and immunofluorescence light microscopy.

    Imaging of polarity during zygotic and somatic embryogenesis of carrot (Daucus carota L.)
    Timmers, A.C.J. - \ 1993
    Agricultural University. Promotor(en): M.T.M. Willemse; J.H.N. Schel. - S.l. : Timmers - ISBN 9789054851004 - 123
    somatische embryogenese - daucus carota - penen - apiaceae - planten - embryologie - cytologie - histologie - immunocytochemie - somatic embryogenesis - daucus carota - carrots - apiaceae - plants - embryology - cytology - histology - immunocytochemistry

    In this thesis a study of the regulation of coordinated growth and the development of polarity during embryogenesis of carrot, Daucus carota L., is described. To this end, several microscopical techniques were used, such as light microscopy, fluorescence microscopy, confocal scanning laser microscopy and electron microscopy. Next to this, immunocytochemical methods were used frequently to localize proteins in plant tissue sections.

    Plants are composed of several types of organs and tissues, each of them having a characteristic structure and function. For the development of a full-grown germling from one cell, the zygote, a tight regulation of growth and differentiation is required. During this process of embryogenesis, growth proceeds through a number of developmental stages which are described subsequently as globular, oblong, heartshaped and torpedo-shaped.

    Despite the large number of observations on embryogenesis, made in various plants, the molecular and cellular basis of this developmental pathway is still poorly understood. The divalent cation Ca 2+participates in the initiation and maintenance of a great variety of physiological processes, including the regulation of cell polarity, cell division, cell growth, cell volume, hormone action and distribution, and enzyme synthesis and activation. Considering the diversity of processes in which Ca 2+is involved, it is to be expected that an investigation of the distribution of Ca 2+, and Ca 2+binding proteins, during plant embryogenesis, will lead to a deeper understanding of the regulation of this process.

    Studies on zygotic embryogenesis are hampered by the presence of surrounding maternal tissue. Therefore, somatic embryos of carrot are used often as experimental substitutes for zygotic embryos, since the discovery of in vitro embryogenesis in carrot cultures in 1958. Carrot somatic embryos can be obtained, relatively easily, in great amounts, essentially free of surrounding tissue, just by transferring cell clusters, designated as proembryogenic masses, from medium supplemented with the growth regulator 2,4-D to medium without 2,4-D. This feature makes carrot an ideal model system for the study of plant embryogenesis.

    In Chapter 1, the general introduction, the zygotic and somatic embryogenesis of carrot is described structurally. Similarities and differences between both processes are mentioned. Many external factors, which are described extensively in the literature, influence the development of somatic embryos. For normal growth and development to occur, the presence of Ca 2+in the medium is absolutely required, and embryogenesis is enhanced specifically by a rise of [Ca 2+]. In this chapter, the role and distribution of Ca 2+in plants is briefly described.

    The principal targets of calcium signals in eukaryotes are calciumbinding proteins of which calmodulin, a protein present in all plant cells, has been studied most extensively. The structure, activity and localization of this acidic, small and heatresistant protein is described from the literature. Chapter 1 ends with a survey of techniques which are nowadays available for the localization of Ca 2+and calmodulin in plants.

    In Chapter 2, chlorotetracycline and fluphenazine, two fluorescent indicators, are being used to localize Ca 2+and activated calmodulin respectively, during carrot somatic embryogenesis. Embryogenesis appears to coincide with a rise in [Ca 2+] and activated calmodulin is mainly found in the future root side of the embryo. It is concluded, that the polarity in the distribution of calmodulin is already present before polarity is visible morphologically.

    Fluphenazine visualizes only activated calmodulin. In Chapter 3, the distribution of both activated and non- activated calmodulin has been studied with the aid of antibodies. Besides the various developmental stages of somatic embryogenesis, also zygotic embryos and a number of stages of zygotic embryo germination have been studied in this chapter. The most striking observation is that the distribution of calmodulin in somatic embryos differs strongly from the distribution in zygotic embryos, but resembles the distribution during zygotic embryo germination. Both in somatic embryos and in germinated zygotic embryos, calmodulin appears to be present mainly in amyloplasts, while in zygotic embryos calmodulin predominantly was found to be localized in the cytoplasm.

    For a detailed analysis of the distribution of Ca 2+in living, intact plant cells in tissues with fluorescent indicators, confocal laser scanning microscopy is the method of choice. A suitable indicator is fluo-3. Unfortunately, the plasma membrane is not permeable for this compound. Therefore, a method had to be developed with which fluo-3 could easily be loaded into plant cells. In Chapter 4, it is described that with the aid of digitonin fluo-3 can be used successfully for the localization of Ca 2+in embryogenic plant cells, in combination with confocal scanning laser microscopy.

    As has already been noticed in Chapter 2, carrot somatic embryogenesis coincides with a rise in [Ca 2+]. In Chapter 5, a detailed analysis has been made of the distribution of free cytosolic Ca 2+during carrot somatic embryogenesis with the aid of the method described in Chapter 4. It appeared that [Ca 2+] is especially high in the protoderm of the embryos, and gradients in [Ca 2+] along the longitudinal axis of torpedo-shaped embryos were frequently observed. Very obvious was the high [Ca 2+] in the nuclei of protoderm cells, This nuclear localization was confirmed by antimonate precipitation, by which Ca 2+is visualized as electron dense precipitates in the electron microscope.

    The concentration of Ca 2+in the cytosol is not only linked with the concentration in the vacuole, but also with the pH of the cytosol and the vacuole. Since both pH and [Ca 2+] are important factors during embryo genesis, in Chapter 6 a study is described of the distribution of pH in vacuoles during somatic and zygotic embryogenesis and during zygotic embryo germination of carrot. Neutral red and acridine orange were used as indicators of vacuolar pH and their distribution has been compared with the distribution of fluphenazine. Strikingly, major similarities were found between the distribution of the three probes used, and all three reacted similarly on treatments with A231 87, EGTA or propionic acid. Confocal microscopy revealed a network of vesicles and tubules, predominantly present in the protoderm of somatic embryos and germinated zygotic embryos after incubation in acridine orange. Proposed is, that calmodulin is possibly involved in the digestion of cell material in autophagic vacuoles or is involved in the regulation of the movements of vacuolar tubules. However, additional research is necessary to explain the observed distribution patterns satisfactory.

    From the previous chapters it appeared that noticeable differences exist between somatic and zygotic embryos in the distribution of anti calmodulin, fluphenazine fluorescence, neutral red and acridine orange. In Chapter 7 a possible structural basis of these differences has been searched for. The distribution of anti-calmodulin, and perhaps also fluphenazine fluorescence, could be linked with the presence of amyloplasts, which were abundant in somatic and germinated zygotic embryos, but which were not found in zygotic embryos. Differences in the localization of neutral red and acridine orange are related to differences in vacuolation between somatic and zygotic embryos.

    Chapter 8 is the general discussion. Here, somatic embryo initiation, its early development and the formation of polarity during somatic and zygotic embryogenesis of carrot are the main topics. It is concluded, that important differences exist between somatic and zygotic embryogenesis and that the process of somatic embryogenesis shows similarities with zygotic embryo germination. The chapter ends with some concluding remarks about the methods used for the localization of Ca 2+and calmodulin.

    Modulation of growth control mechanisms critical to atherogenesis
    Zwijsen, R.M.L. - \ 1992
    Agricultural University. Promotor(en): J.H. Koeman; R.C. van den Bos. - S.l. : Zwijsen - ISBN 9789054850342 - 125
    atherosclerose - arteriën - celdeling - voortplanting - celcyclus - cytologie - atherosclerosis - arteries - cell division - reproduction - cell cycle - cytology

    The principal lesion characteristic of atherosclerosis is the plaque. This lesion mainly consists of smooth muscle cells, connective matrix and large amounts of extracellular lipids. Smooth muscle cell hyperplasia is an integral event in atherosclerotic plaque formation and the resultant occlusion of blood vessels. These abnormal cell proliferations are primarly caused by defects in the autocrine and/or paracrine growth regulation. A novel mechanism critical to atherogenesis introduced in the present study is gap junctional mediated growth control. The atherogen-induced effects on gap junctional communication between human smooth muscle cells are described in chapters 2,3 and 4. The role of autocrine growth stimulation in atherogenesis is shown by the demonstration of the presence of activated transforming genes in atherosclerotic lesions (chapter 5) and the cell transforming potential of (atherogenic) low density lipoproteins (LDL) as shown in chapter 6. Chapter 5 also demonstrated that several transforming genes may be present in human plaque DNA, one of them being PDGF-A (platelet-derived growth factor chain A). The expression of the latter gene can be modulated by oxidized LDL (chapter 7).

    The present investigation provides additional evidence for the hypothesis that autocrine production of growth modulating factors may contribute to the characteristic abnormal smooth muscle growth in atherogenesis. We demonstrated that lipoproteins with atherogenic potential, such as LDL, in contrast to the non-atherogenic HDL, are able to transform fibroblasts in vitro. Genetical alteration of intimal cells present in atherosclerotic lesions was demonstrated using DNA-mediated transfection techniques. It could be demonstrated that human aortic plaque DNA contains active transforming genes, as PDGF-A. This is in agreement with studies by Penn et al. (1986) and Ahmed et al. (1990), showing transforming genes in human coronary artery plaques. Even DNA of cultured human atherosclerotic plaque cells appeared to have cell transforming capacities (Parkes et al., 1991). Only Yew et al. (1989) could not demonstrate a transforming potential of human plaque DNA. The variable results obtained sofar might indicate that different mechanisms are involved. It should be stressed in this respect that in all these studies pooled plaque samples were used and it should be realized that the transforming potential is not necessarily present in each plaque. The observed transforming potential of plaque DNA supports very well the monoclonal hypothesis of Benditt and Benditt (1973). They observed that atherosclerotic lesions start as singular focal masses containing monoclonality and suggested that these masses were in general similar to benign tumors as observed in other tissues. However, it has been argued that apparent monoclonality could also arise because of strong selection pressure favoring a subpopulation of cells, although findings of transforming genes, such as PDGF-A, present in atherosclerotic lesions and the transforming potential of atherogens indicate the opposite. Additional arguments in favor of the monoclonal hypothesis are that tumor initiators like chemical mutagens and promutagens (Albert et al., 1975; Bond et al., 1981), radiation (Gold, 1961) and oncogenic viruses (Minick et al., 1979) can induce atherosclerotic lesions in laboratory animals. Hence, molecular alterations underlying the proliferation of smooth muscle cells could show resemblance to the molecular events, which are critical in the development of cancer.

    The current view on atherosclerosis is based on the "response to injury" hypothesis, implying a paracrine stimulation of smooth muscle growth as a result of injury of endothelium (Ross, et al., 1976; Ross, 1981). Most of the studies of experimental atherogenesis are implemented in the framework with this hypothetical model. Support for this hypothesis is based on the following observations. Atherosclerotic lesions can be induced in experimental animals by endothelial denudation and factors derived from platelets can induce smooth muscle growth in vitro. Injury to arterial endothelium by mechanical, chemical, toxic, viral, or immunological agents caused endothelial denudation, and was followed by platelet adhesion and aggregation, with consequent release of platelet-derived growth factors (PDGF), in turn leading to migration into and proliferation of smooth muscle cell in the intima and secretion of connective tissue components. Over the years, the hypothesis has been modified (Ross, 1990). First of all, it became apparent that actual denudation was not a consistent early feature of atherosclerosis (Davies et al., 1976). Secondly, platelet adherence is neither necessary nor sufficient to cause the lesions (Schwartz and Reidy, 1987). Presently, the view is: a. the endothelium can respond to a variety of stimuli, by subtle changes in function (endothelial dysfunction) and/or by the induction of new endothelium properties; thus non- denuding injury may be important in initiating the lesions of atherosclerosis and b. platelets are not the sole initiators of smooth muscle proliferation, since growth promoting and growth inhibitory factors secreted by other cell types, including macrophages and endothelial cells, may modulate smooth muscle growth.

    However, not all observations made can be explained by the "response to injury" hypothesis, including the findings that (intimal) smooth muscle cells themselves can secrete and can respond to growth modulating factors and the cell transforming potential of plaque DNA as demonstrated above, thus implying an involvement of autocrine growth stimuli. A good candidate for autocrine growth factor stimulation of smooth muscle cells is PDGF-A. High transcript levels of this transforming gene were detected in intimal cells of human atherosclerotic lesions (Libby et al., 1988; Wilcox et al., 1988). The present study showed that PDGF-A was identified as one of the transforming genes present in human plaque DNA and the expression of this gene can be induced by LDL (oxidized). HDL, a protective lipoprotein to atherosclerosis, did not modulate PDGF-A transcript levels in smooth muscle cells. While both oxidized LDL as well as PDGF-A transcripts are detected in atherosclerotic lesions this molecular mechanism could play a role in atherogenesis.

    Furthermore, the present study provides strong evidence that disturbance of intercellular communication is another mechanism, which is probably important in the etiology of atherosclerosis. It is shown that compounds with atherogenic potential inhibit communication between human smooth muscle cells. The potency to modulate cell-cell communication correlates well with their atherogenic potential. For example, low density lipoproteins (LDL) in oxidized form and cholesterol oxidation products inhibited cell-cell communication. A non-atherogenic lipoprotein as high density lipoprotein (HDL) did not influence gap junctional communication between human smooth muscle cells. Otherwise, growth factors like PDGF and EGF can modulate cell-to-cell communication (Maldonado et al., 1988; Madhukar et al., 1989). This would imply that both paracrine and autocrine growth factor production are involved in atherogenesis, in which their influence on the gap junctional mediated growth control mechanism is a vital step in atherogenesis. At this stage, disruption of intercellular communication results in an uninhibited multiplication of these cells leading to the formation of (monoclonal) atherosclerotic lesions.

    The main conclusion of the present study is that the three phenomena
    1) production of growth modulating factors by vascular cells, including endothelium, monocytes, platelets and smooth muscle cells
    2) DNA modification (e.g. PDGF-A) of smooth muscle cells
    3) loss of intercellular communication
    together are important factors in the process of atherosclerosis. The two main theories on atherosclerosis, the "monoclonal" and the "response to injury" theory, are compatible in many respects and can be fit into one unifying hypothesis as described in Fig. 8.1

    The figure shows that one of the earliest events in atherosclerosis is an increased adhesion of monocytes to what appears to be intact arterial endothelium, a phenomenon which can be well demonstrated using scanning electron microscopy (Faggiotto et al., 1984). In this process lipid factors like hypercholesteraemia and other factors as IL-1 or TNF (which introduces adhesion molecules. on endothelial cells) are important triggers. As a consequence of monocyte emigration, there is focally increased permeability to LDL and macromolecules (Territo et al., 1984; Gerrity et al., 1979). An altered endothelial function (dysfunction) causing increased permeability may also be induced by risk factors such as hypertension, hyperlipidaemia, smoking, immunological factors, stress and diabetes mellitus (Reidy, 1985; Sieffert et al., 1981; Gordon et al., 1981, Munro and Cotran, 1988). In this way the agents present in the bloodstream (such as LDL) accumulate and are modified in the arterial wall and thereafter exert their effects locally on vascular cells.

    The next important step could be DNA modification(s) of smooth muscle cells as shown by the presence of transforming genes, as PDGF-A, in lesion DNA and the effects of the atherogenic LDL on cell transformation. This defect of an autocrine growth regulation is followed by another vital growth control mechanism, the gap junctional communication. Communication between human smooth muscle cells has been modified by several atherogens, like LDL (oxidized) and oxysterols. In case of impairement of both growth regulation mechanisms clonal growth may occur.

    The processes described on the left side of the scheme are consistent with the paracrine proliferation mechanism of "the response to injury" theory of Ross (1990). The first trigger in this process is a dysfunction of the endothelium. This process introduces the adhesion and aggregation of platelets and/or monocytes to the endothelium, which results in a release of growth modulating factors as platelet-derived growth factor. At this stage, paracrine growth stimulating factors do not yet cause abnormal smooth muscle growth, because autonomous growth of cells still is controlled by gap junctional communication with surrounding cells. Only when growth factors (and atherogens) inhibit gap junctional communication abnormal smooth muscle cell proliferation occurs. Thus, inhibition of gap junctional intercellular communication could play a predominant role in the onset of atherogenesis.

    A better understanding of these growth modulating processess will improve the possibility to study the possible atherogenic properties of chemicals within the framework of toxicological research.

    Immunofluorescence microscopy and dilution-plating for the detection of Xanthomonas campestris pv. campestris in crucifer seeds : methods to determine seed health and seed infection
    Franken, A.A.J.M. - \ 1992
    Agricultural University. Promotor(en): J. Dekker. - S.l. : Franken - 165
    plantenziekten - plantenziekteverwekkende bacteriën - koolsoorten - kleuring - fluorescerende kleurstoffen - immunologische technieken - elisa - xanthomonas - cytologie - histologie - immunologie - antigenen - plant diseases - plant pathogenic bacteria - cabbages - staining - fluorescent dyes - immunological techniques - elisa - xanthomonas - cytology - histology - immunology - antigens
    Black rot is one of the most threatening diseases of crucifers. The causal agent of this disease is the bacterium Xanthomonas campestris pv. campestris . The bacterium attacks all cultivated brassicas, radishes and numerous weeds, and is able to survive on plant debris in the soil. The primary source of inoculum is often infected seed. The most important ways to control black rot are the use of resistant cultivars and the use of 'healthy' seed. To obtain healthy seed, chemical or physical seed treatments may be used. These seed treatments may, however, seriously damage the seed quality (germination), may cause phytotoxicity or may not sufficiently eliminate the black rot pathogen from the seed (chapter 2). To check the seed health, sensitive and specific methods are needed to detect X. c. pv. campestris in the seed. Several methods for detecting X. c. pv. campestris are summarized in chapter 2. The most commonly used assays are plating assays, in which seed washings are plated onto isolation media, such as BSCAA (basal starch cycloheximide agar with nitrofurantoin and vancomycin), CS20ABN (a starch medium with bacitracin, neomycin and cycloheximide), FS (a medium with starch trimethoprim, cephalexin, cycloheximide, methyl green), NSCA (nutrient starch cycloheximide agar), NSCAA (NSCA with nitrofurantoin and vancomycin), and SMA-medium (starch-methionine agar with cephalexin and nitrofurantoin). Serological assays such as enzyme immuno-assays, Ouchterlony double diffusion, agglutination and immunofluorescence microscopy (IF) can be used for identification of pure cultures of X. c. pv. campestris . Cross-reactions with other pathovars of X. campestpis have, however, been reported. So far, IF using polyclonal antisera was the only serological technique employed for detecting X. c. pv. campestris in seed washings.

    The aim of this study was to analyse important characteristics of IF and plating assays, and to improve their use for identification and detection of X. c. pv. campestris in crucifer seeds. Methods for the detection and identification of X. c . pv. campestris are reviewed in chapter 2. In chapter 3 some aspects of plating assays for isolation of Xanthomonas campestris pv. campestris from crucifer seeds are discussed. Little differences were found between results obtained with NSCA, NSCAA and FS medium. It was, however, noted that the performance of the media often depends on the seed lot and extraction method used. Therefore, probably other methods such as immunofluorescence microscopy (IF) are needed to confirm presence of X. c. pv. campestris with higher certainty. With the extraction methods 2.5 h shaking and 1.5 h soaking, more colonyforming units were recovered from some seed lots than with the standard 5 min shaking of seed lots. However, prolonged extraction did not result in finding more seed lots infected. However, the use of two methods for extracting X. c. pv. campestris from crucifer seed, will enhance the chance of isolating the pathogen from the seed (chapter 3 and 6).

    In chapter 4 the specificity of polyclonal antisera and monoclonal antibodies for identification of X. c. pv. campestris is discussed. Polyclonal antisera reacted in IF with all strains of X. c. pv. campestris and other xanthomonads (e.g. X. c. pv. vesicatoria and amoraciae ) at low dilution (1:100). Non-xanthomonads also reacted with 2 out of 3 polyclonal antisera at this dilution. At higher dilutions (1:900), however, most crossreactions with non-xanthomonads disappeared as well as reactions with some strains of X. c. pv. campestris and other pathovars. Six monoclonal antibodies (MCA 17C12, MCA 16B5, MCA 20H6, MCA 2F4, MCA 18G12, MCA 10C5), produced against X. c. pv. campestris were tested in immunoblotting (IB), an enzyme immunoassay (EIA), dot-blot immunoassay (DBI) and IF. The monoclonal antibodies reacted with the lipopolysaccharide (MCA 20H6, 2F4, 18G12, and IOC5) or membrane proteins (MCA 17C12 and 16B5) of X. c. pv. campestris in IB Two monoclonal antibodies (MCA 17C12 and 16B5) reacted with all xanthomonads tested in DBI, but not in IF and EIA. The other monoclonal antibodies (MCA 20H6, 2F4, 18G12, and 1OC5) did not react with all strains of X. c. pv. campestris and did react with some other xanthomonads, such as X. c. pv. vesicatoria and amoraciae, in IF, EIA and DBI. It was concluded that some polyclonal antisera and monoclonal antibodies may be used for identification of (a group of) strains of X. c. pv. campestris. The question, whether monoclonal antibodies, as compared to a polyclonal antiserum, may give rise to false- negative or false-positive results when testing seed lots, is dealt with in chapter 5. IF with one polyclonal antiserum
    and monoclonal antibodies was used for direct detection of X. c. pv. campestris in seed washings and identification of colonies. For direct detection of X. c. pv. campestris in seed washings, it was found that IF-results (cell counts) depend in part on the seed lot tested, the extraction method and antibodies used. This confirmed the interactions found in plating assays. The polyclonal antiserum tested (PCA 94) did not always result in detection of more fluorescent cells in IF. On the contrary, for some seed lots and extraction methods the opposite was found; monoclonal antibodies sometimes detected more fluorescent cells than polyclonal antisera. When using two extraction methods (5 min and 2.5 h shaking at room temperature) for one seed lot the risk of false-negative results in IF could be minimized (chapter 5 and 6). When monoclonal antibodies were used for identification of colonies, it was found that some X. c. pv. campestris colonies did not react with antibodies MCA 16B5, MCA 17C12, and to a lesser extent with MCA 1OC5, MCA 2F4, and MCA 18G12. Saprophytic isolates of one seed lot cross-reacted with MCA 17C12 and to a lesser extent with MCA 2F4, 18G12 and PCA 94. On the basis of this study MCA 20H6 was considered to be the most suited antibody for detection and identification of X. c. pv. campestris .

    Chapter 6 shows that the correlation between IF and dilution-plating was similar for one monoclonal antibody (MCA 20H6) and one polyclonal antiserum tested (PCA 94). With increasing cell numbers in IF the chance of isolating X. c. pv. campestris also increased. With IF generally much more seed lots were found positive than with dilution-plating. It was shown that the correlation between IF and dilution-plating depended in part on the volume of seed extract examined. When examining relatively large volumes (e.g. 50 μl), the sensitivity of IF will be enhanced and the risk of false-negative reactions in IF as compared to dilution-plating will be decreased. On the other hand the correlation with dilution-plating decreased. It was concluded that IF generally gives a good prediction of 'health' of a seed lot and that dilution-plating generally gives a good prediction of 'disease'. Chapter 7 summarizes the problems with serological techniques for seed-borne bacteria and gives possible solutions to be worked out in future.

    To minimize the risk of false-negative results, it is advisable to use at least two plating media (e.g. NSCAA and CS20ABN) in dilution-plating and two extraction methods (5 min and 2.5 h shaking at room temperature) in both IF and dilution-plating. For IF, it is advisable to screen either with different monoclonal antibodies separately, with a mixture of monoclonal antibodies (e.g. MCA 20H6, MCA 2F4 and MCA 18G12), or both a monoclonal antibody (MCA 20H6 or 2F4) and a high quality polyclonal antiserurn (PCA 94).

    More research is needed, however, to assess the significance of interference by saprophytes and antibiotic substances, released by the seed or saprophytes, with the detection assay and the survival of the pathogen in the seed.

    Isolation and characterization of Spinacia oleracea L. sperm cells
    Theunis, C.H. - \ 1992
    Agricultural University. Promotor(en): J.L. van Went; H.J. Wilms. - S.l. : Theunis - 89
    spinacia oleracea - spinazie - chenopodiaceae - gameten - spermatozoön - eicellen - cytologie - histologie - spinacia oleracea - spinach - chenopodiaceae - gametes - spermatozoa - ova - cytology - histology

    Gametes are specialized cells with the natural capacity to fuse in a well determined way. The fusion products are intended to develop into new individuals. Basic knowledge of gametes is of great importance for both traditional plant breeding as well as for modem biotechnology and gene manipulation. For applications in these fields, more knowledge is necessary of the characteristics of gametes and the mechanisms involved in the process of gamete recognition and fusion. Isolated gametes form ideal material to investigate this. The present study was focused on the isolation and characterization of the male gametes, the sperm cells.

    In chapter 1 an introduction is given. The current information we have on in situ sperm cells, and on the subject of sperm cell isolation, is summarized in this chapter.

    In chapter 2 the ultrastructure is described of the pollen grains of Spinacia oleracea and the sperm cell pair therein. The pollen grain is trinucleate and consists of a vegetative cell and two sperm cells. The pollen grain wall is tectate, with many germination pores, which have a hexagonal distribution. The vegetative nucleus together with the sperm cells are located in the periphery of the pollen grain and are organized in a "male germ unit". The cytoplasm of the vegetative cell contains vacuoles and electron dense vesicles. The mitochondria have a size of 0.3 μm to 0.5 μm. The ER is often organized in single elements, and bears ribosomes. The plastids are filled with starch and only the outer membrane is visible. The high amount of starch may be used in an autotrophic way of germination, or for osmotic stabilization during germination. Microtubules are not found in the vegetative cytoplasm.

    The sperm cell inside the pollen grain contains a heterochromatic nucleus, mitochondria, dictyosomes, and ER. The two sperm cells are attached to each other. They form a pair which is surrounded by a vegetative plasma membrane. Only a few microtubules have been shown in the sperm cell cytoplasm. In previous studies, microtubules have been clearly demonstrated inside the sperm cells. Therefore, it was concluded that the used method of freeze substitution does not completely stop the breakdown of microtubules.

    In order to release the sperm cells, the vegetative cells has to be opened. The osmotic shock method, which has been used in some species, does not work for pollen grains of Spinacia oleracea. Even in pure water only a small percentage of the pollen grains bursts. For this reason, a mechanical method has been developed, using a glass roller to squash large quantities of pollen grains. This method is described in chapter 3.

    Because of the squashing of the grain, the sperm cells are released from the pollen grain together with most of the vegetative cytoplasm. Since physical breaking is applied, any medium can be chosen in which the breaking is performed. After the squashing, the mixture of pollen grains, free sperm cells, vegetative nuclei, vegetative organelles, and pollen grain fragments is filtered over a 25 μm nylon filter. Subsequently, the filtrate is centrifuged on a 20% percoll layer for further elimination of small debris. With this method, a fraction is obtained which contains numerous sperm cells, but which is still contaminated with small vegetative organelles, and small pollen fragments. The yield is approximately 5-10% with a final concentration of 4x10 6sperm cells/ml.

    The free sperm cells are elongated just after squashing, but become spherical after a short time. The originally paired sperm cells separate. The close association of the sperm cell pair with the vegetative nucleus is not maintained during squashing and is therefore, not a firm binding. The sucrose concentration of the medium does not influence the change in shape of the free sperm cells. The volume, however, is influenced by the osmotic value of the medium. The 25 % sucrose concentration was chosen for the rest of the experiments in order to avoid damage caused by osmotic swelling. The diameter of the isolated sperm cells can vary from 4 μm to 9 μm, depending on the sucrose concentration.

    Immediately after isolation, more than 90% of the sperm cells is viable (tested with the fluorescein diacetate test). Soon after isolation, however, some of the cells loose their viability. After 18 h, only 50% of the isolated cells is still viable. Storage of the isolated sperm cells at low temperatures (0°C) doubles the lifespan. Addition of 1 % vitamin C also enlarges the lifespan. It is concluded that depletion of energy is not the cause of the loss of viability, since addition of 0.1 M ATP makes no difference for the lifespan.

    With histochemical tests, using calcofluor white MR2 for cellulose, analine blue for callose, and the PAS reaction for carbohydrates, no cell wall material was observed around the isolated sperm cells. This indicates that the cells are true protoplasts.

    In chapter 4 the results are presented of the analysis of the numbers of mitochondria in isolated sperm cells. To visualize the mitochondria, two staining methods have been used. The DiOC 6 (3) staining (in a concentration of 0.1 μg/ml) gives better results with less background staining, than the Rhodamine 123 staining.

    The analysis was carried out on individual sperm cells, as well as on sperm cell pairs. If individual sperm cells were used, two populations of sperm cells seemed to be present, with an average of respectively 10.3 and 17.8 mitochondria per sperm cell. However, by counting the mitochondria in sperm cell pairs, it is found that there is only one population of sperm cells. The average is 12.4 ± 4.6 mitochondria per sperm cell. The number of mitochondria per sperm cell varies from 2 to 25, which is a high variation. This high variation can be explained in two ways. It is possible that already after the division of the microspore, a high variation exists in number of mitochondria per generative cell. The second explanation can be, that during the development of the generative cell and/or sperm cells, mitochondria are produced or lost.

    With the technique of freeze-fracturing, the plasma membranes of the isolated sperm cells were examined, including the intra-membrane particles (IMP's). In chapter 5 the results are presented. Also with this method, no remnants of the vegetative plasma membrane were found around the sperm cells. Only incidentally, transverse fracture planes through whole sperm cells are found. Most of the fracture planes of sperm cells follow the sperm cell plasma membrane exposing either the PF or the EF face. Neither the ES or the PS faces are found. Both the PF as well as the EF face show IMP's. These IMP's are randomly distributed, and no pattern can be recognized. The PF face has a density of 719 IMP's/μm 2. The EF face has a density of 2088 of IMP's/μm 2. Evidently, the EF half of the sperm cell membrane contains approximately 3 times more IMP's than the PF half. For sporophytic protoplast it has been reported that the PF half contains more IMP's than the EF half. The specific IMP distribution in the sperm cell plasma membrane may be related to the process of gamete recognition and subsequent fusion. With respect to IMP's density, only one type of sperm cells was observed and therefore for this character, no dimorphism could be established.

    Morphometrical and ultrastructural characterization of the isolated sperm cells has been reported in chapter 6. Experiments with various fixations demonstrate that sperm cells are fragile, and difficult to fixate. The osmotic value of the fixation media appear to be of great importance. Also with this method it is clear that the isolated sperm cells are separate and completely spherical. The surrounding vegetative plasma membrane has disappeared. No cell wall material is observed. The sperm cell contains a large nucleus which can be either heterochromatic or euchromatic. The mitochondria are spherical and frequently appear to be clustered in groups of 5 to 10 mitochondria, but also individual mitochondria have been observed. The dictyosomes have 4 to 5 cisterns with associated small vesicles. Small vacuoles are present. The endoplasmatic reticulum is sparse, and often dilated. Ribosomes are sometimes grouped in polysomes. No microtubules have been observed. From these observations it is clear that isolated cells contain a similar set of organelles as the in situ sperm cells. After measuring the section diameters, the average diameter of the complete cell is calculated to be 3.66 μm with the used fixation conditions. From surface area's in the sections is calculated that 50 % of the sperm cell is occupied by its nucleus, 2.5 % of the cell is mitochondria, and 0. 6 % of the cell is dictyosome. The ultrastructural analysis did not give any indication that in Spinacia oleracea sperm cell dimorphism in regard of sperm cell size, exists.

    In chapter 7 the results of the present thesis are discussed in general sense and in a broader context. Results which have already been discussed in previous chapters are not further discussed in this general discussion.

    The isolation technique of "physical breaking", developed for Spinacia oleracea is compared with the "osmotic shock" technique, used for other species. The advantages and disadvantages of both techniques are presented and discussed. The usefulness of the two techniques in further research of isolated gametes are explained. Preferences for one of the two technique clearly depends on the aims of the further research.

    The general discussion highlights the two major phenomena sperm cells show when isolated: the changing of a spindle shaped cell to a spherical cell, and the loss of viability. The change in shape occurs in all species observed thus far. Likely, the change in shape is a natural process which also occurs during fertilization. The loss of viability can be slowed down with low temperature, and anti-oxidantia. During the natural fertilization process, extended viability is not necessary, since the free sperm cells fuse rapidly with the female partners. So also for this character it may be a natural phenomenon.

    Sperm cell dimorphism, which has been reported in some species, is not found in Spinacia oleracea. The isolated sperm cells form good material to study dimorphism because of the large ~ties of cells which can be analyzed.

    Cell fate establishment during early development of cyprinid fishes, with special emphasis on the formation of the primordial germ cells
    Gevers, P. - \ 1992
    Agricultural University. Promotor(en): L.P.M. Timmermans; H.W.J. Stroband. - S.l. : Gevers - ISBN 9789054850373 - 129
    cyprinidae - karper - eieren - ontwikkeling - cytologie - celbiologie - cyprinidae - carp - eggs - development - cytology - cellular biology

    Cell fates can be established either by preformation or by epigenesis. With respect to primordial germ cells (PGCs) it has been shown that the Amphibia exhibit both types of cell fate establishment. Therefore, it is important to study the germ cell origin of the evolutionary lower class of fishes.

    In order to study the origin and cell fate establishment of a specific cell type, two approaches are possible. Firstly, its morphological characteristics may be studied, and traced back to the earliest possible developmental stage. Using this approach, we studied in Barbus conchonius, a cyprinid fish, the presence, location and morphology of cells, containing nuage, an ultrastructural electron dense perinuclear material, generally accepted to be characteristic for germ cells (Chapter 2). The results show that nuage containing cells, PGCs, could be found from 10 h after fertilization (a.f.) onwards (around 100% epiboly). They translocate between 10 h and 12 h a.f. from a position within the mesoderm towards a position between mesoderm and yolk syncytial layer (YSL). However, PGCs remain separated from the YSL by extensions of endodermal cells. The location of PGCs at the stage of first identification may indicate that they originate within the mesoderm. Contacts with endodermal cells may be involved in the formation of their characteristics.

    A second approach for studying the origin of PGCs concerns the labeling of blastomeres during cleavage stages and following their progeny up to the stage of morphological recognition of PGCs (Chapter 3). Using this method, a cell lineage tracer, Lucifer Yellow - Dextran (LY-D), was injected into individual blastomeres of the 64 cell stage of Barbus conchonius embryos. Study of the fate of their progeny revealed that a lower layer cell (LLC) could give rise to both somatic cells and a number of PGCs (in about 25% of the cases), while injections into upper layer cells (ULC) only resulted in labeled somatic cells. The distribution of somatic progeny after injection of a certain blastomere was unpredictable with respect to both the later location within the embryo and the tissue type of its descendants. Unpredictable cell lineages were probably due to extensive intermingling of cells during epiboly. The results suggest that the formation of PGCs as well as that of somatic cells is an epigenetic process. As mentioned above, the PGCs may originate within the mesoderm (Chapter 2).

    Since in Barbus conchonius cells apparently are not yet committed to their fate during cleavage and start extensive intermingling during epiboly, the question arose when cell fate restrictions occur (Chapter 4). These restrictions may also be important for the determination of a diversity of cell types, including PGCs. Changes in communication properties between cells or cell groups may be correlated with differences of their developmental pathways. Since it was known that during early epiboly certain deep cells are dye-coupled to the yolk syncytial layer (YSL), injections of Lucifer Yellow (LY) into the YSL at several stages of epiboly were used in order to study changes in communication properties. The formation of a group of dye-coupled cells, indicating a group of gap junctionally communicating cells, was described. At the onset of epiboly LY appeared to be transferred from the YSL to all blastodermal cells. Between 40% and 60% epiboly we observed a ringshaped group of labeled cells, which probably had involuted during early gastrulation. This cell group correlated with the leading edge of the hypoblast, and was dye-uncoupled from the uninvoluted epiblast. From 60% epiboly onwards the blastodermal cells were dye-uncoupled from the YSL. Between 50% and 100% epiboly the ring-shaped labeled hypoblast was, due to convergent cell movements, gradually transferred towards a dorsally located bar-like structure. Gap junctions appear to connect cells with the same fate. Consequently, the appearance of part of the hypoblast as a dye-coupled cell layer, which is dye-uncoupled from the epiblast, may correlate with early restriction in cell fate. On the other hand, gap junctions between YSL and DC may be involved in the transfer of an inducing signal at the onset of gastrulation.

    Apparently, it is only from gastrulation onwards that developmental pathways of cell groups gradually disperse. Since cell determination appeared to be highly regulative, knowledge of the control of the directional mass cell migration during gastrula stages may be important for understanding cell fate establishment. Because fibronectin (FN) appeared to be involved in the guidance of migrating cells in amphibian embryos, the presence of FN during epiboly and gastrulation was studied in embryos of the common carp ( Cyprinus carpio ; Chapter 5). However, in order to establish that FN is also involved in early fish development, we first performed a pilot study in which the functional role of FN was blocked by treatment with GRGDS, interfering with interactions between FN and its receptor. Since this treatment resulted both in retardation of epiboly, and a lack of involution, it became interesting to study the localization of FN during respective stages. Using immunocytological methods, FN first appeared present on a number of cell membranes during early epiboly. During the progress of gastrulation (from 50% epiboly onwards) we observed a gradually increasing number of epiblast
    cells, which were labeled on their YSL facing surface. They were first present in front of the migratory hypoblast cells and later partly separated the hypoblast cells from the epiblast. The results suggest that also in cyprinid fishes FN may be involved in the guidance of gastrulating cells.

    In conclusion, PGCs in embryos of cyprinid fishes can be recognized from late gastrula stages onwards by the presence of nuage. They are supposed to arise epigenetically from the mesoderm, possibly by inducing influence of endodermal cells. Determination processes of both PGCs and somatic cells probably do not start until early gastrulation (50% epiboly). FN may be involved directly (induction processes) or indirectly (morphogenetic movements resulting in induction) in epigenetic processes.

    A histochemical study of root nodule development
    Wiel, C. van de - \ 1991
    Agricultural University. Promotor(en): A. van Kammen. - S.l. : Van de Wiel - 189
    wortelknolletjes - knobbelvorming - histologie - cytologie - root nodules - nodulation - histology - cytology

    In cooperation with soil bacteria of the genera Rhizobium , Bradyrhizobium or Azorhizobium , many members of the legume family are able to form specialized organs on their roots, called root nodules. The bacteria, wrapped up inside a plant membrane, are accomodated in large parenchymatic cells located centrally in these root nodules. For this, they reward their host by converting atmospheric nitrogen into a form usable for the plant. The central infected tissue of the nodule is surrounded by a peripheral tissue provided with vascular bundles through which metabolites are exchanged with the other parts of the plant.

    In the interaction with the bacteria, the host plant expresses specific genes that are not transcribed at a detectable level in other parts of the plant. The products of several of these genes are made during the formation of the nodule and are named early nodulins.

    The present study aims at elucidating the role of these early nodulins in the formation and infection of the root nodules. For that purpose, we set out to combine the molecular approach of studying gene expression with the microscopical approach of studying the structural development of the nodule.

    To provide a background to these studies, chapter 11 summarizes existing knowledge about nodule development from an anatomical/cytological point of view, supplemented with data on already described nodulins and with brief excursions into physiological phenomena relevant to the rest of our study.

    In chapter III and IV, nodulin gene expression is analysed in common vetch ( Vicia sativa ) nodules elicited by a panel of bacterial strains with various defined genetic changes. Such nodules were blocked at different stages in the development of the central tissue depending on the bacterium involved; the precise stage at which the blockade occurred was determined by light- and electron-microscopical observations. In that way, insight could be gained in the diverse genetic information supplied by the bacterium for nodule development to proceed through the successive developmental stages and the induction of the appropriate nodulin genes going with it. Furthermore, the start of the expression of individual nodulin genes, for instance the early nodulin Nps-40', could be related to certain stages of central tissue development. In the case of the leghemoglobin genes, such a correlation between nodulin gene expression and specific developmental stages could be confirmed by the direct localization of the leghemoglobin proteins in pea ( Pisum sativum ) nodule sections comprising different consecutive developmental stages, by immunolabeling.

    Such direct approach of studying nodulin gene expression in nodule sections was further pursued in the chapters V, VI, VII and VIII. In chapters V, VI and VII early nodulin gene transcripts for which sequenced cDNA clones had become available were localized by in situ hybridization: in chapter V, ENOD2 in soybean ( Glycine max ) and pea nodules, respectively; in chapter VI, ENOD2 in alfalfa ( Medicago sativa ) nodules; and in chapter VII, PsENOD12 in pea. In chapter VIII an attempt to localize the Nps-40' protein by immunolabeling in pea nodules is described. By these in situ localization methods, different temporal and spatial patterns of gene expression for each early nodulin were determined. Speculations about the functions of the individual nodulins are made based upon the gene expression patterns and the amino acid sequences of the nodulins as deduced from the nucleotide sequence of the corresponding cDNA clones.

    In addition, in chapter VI, in situ localization of MsENOD2 transcripts was performed on alfalfa nodules induced by certain engineered bacterial strains or by auxin transport inhibitors. Such nodules do not have bacteria in their central tissue and also differ in other structural details from effective nodules, but nevertheless were shown to exhibit a tissue- specific expression pattern of the MsENOD2 gene similar to effective nodules. In chapter VII the results of further experiments are reported pertaining to the influence of the bacterium on nodulin gene expression, particularly the involvement of bacterial factors and the bacterial nod genes in the induction of the expression of the PsENOD12 genes.

    Finally, chapter IX summarizes the results of the in situ localization of early nodulin gene products. In the light of these results, the significance of our histochemical approach to elucidating the role of nodulins in root nodule development is discussed.

    Induction and characterization of micronuclei in plant cells : perspectives for micronucleus-mediated chromosome transfer = Inductie en karakterisering van microkernen in plantecellen : perspectieven voor chromosoom overdracht via microkernen
    Verhoeven, H.A. - \ 1989
    Agricultural University. Promotor(en): A. van Kammen; B. de Groot. - S.l. : Verhoeven - 117
    cytologie - plantenfysiologie - genetische modificatie - recombinant dna - celfysiologie - cytology - plant physiology - genetic engineering - recombinant dna - cell physiology

    In this thesis, micronucleation in plant cells has been investigated and systems for isolation and transfer of organelles have been established.
    The discovery, described in chapter two, that the phosphoric amide herbicide amiprophos-methyl induced micronuclei at a high frequency in cell suspensions of N.plumbaginifolia, has opened the possibility to develop a microcell-mediated chromosome transfer system analogous to that in mammalian cell lines. In mammalian cells, micronuclei are induced by prolonged exposure of cells to spindle toxins (colchicine, Colcemid), resulting in up to 60% micronucleated cells (Matsui et al., 1982). Micronucleated cells are isolated by the "shake-off' method, and subjected to high speed centrifugation, which results in fractionation of the cells into microcells, containing micronulei with one or a few chromosomes. Subsequently, microcells are fused to the recipient cells. The transferred chromosomes were found to remain intact and mitotically stable (Fournier, 1982). This technique has hitherto not been available for plant cells or protoplasts, due to the lack of efficient procedures to induce micronuclei. Gamma-irradiation is now often used in the construction of monochromosomal addition lines by somatic hybridization (Bates et al., 1987), to induce chromosome damage which promotes chromosome elimination from one of the fusion partners. As has been pointed out in the introduction (chapter one), ionizing radiation induces chromosome rearrage ments, deletions and insertions (Menczel et al., 1982). From research on mammalian cells, it is known that these phenomena occur with a lower frequency after microcell-mediated chromosome transfer (Fournier, 1981). If microprotoplasts would become available for plant genetic manipulation, transfer of a limited number of chromosomes by microprotoplast fusion would offer an alternative to the use of gamma-irradiation. With the finding that APM induces micronuclei at high frequency in plants, transfer of low numbers of chromosomes after micronucleation can now be tested for use in plant genetic manipulation. The APM treatment was found to be reversible, as was demonstrated by washing the cell suspension cultures free from APM. After washing, normal growth and cell division were soon resumed, with some abnormal, multipolar spindles in the first division after washing. This observation is in good agreement with the the reversible inhibition of microtubule polymerization by APM (Falconer and Seagull, 1987). This low cytotoxicity makes APM a useful tool in the induction of micronuclei in plants.

    The flow cytometric analysis of the nuclear DNA content of APM-treated cel suspension cultures of N.plumbaginifolia, revealed the presence of many micronuclei with a DNA content equivalent to one metaphase chromosome (which consists of both sister chromatids). Similar observations have been made in micronucleated rat kangaroo cells after treatment with Colcemid (Sekiguchi et al., 1978). Sorting of the micronuclei on the basis of the fluorescence of ethidium-bromide, followed by analysis of the DNA content by Feulgen staining (chapter three), shows that it is possible to separate micronuclei on the basis of their DNA content by flowcytometry, like it has been shown for isolated plant metaphase chromosomes. Chromosome identification is sometimes possible with isolated metaphase chromosomes (de Laat and Blaas, 1984; Conia et al., 1987a; 1987b). Identification of chromosomes present in a particular micronucleus is not possible. This is due to different degrees of chromosome decondensation in the micronuclei (which influences the fluorescence signal of the fluorochrome -DNA complex by quenching), and due to the various combinations of chromosomes in micronuclei containing more than one metaphase chromosome. This is illustrated by the DNA histograms of isolated micronuclei in chapter two, which lack the specific chromosome peaks, present in metaphase chromosome preparations (chapter four). When micronuclei are present in large numbers, the overall DNA histogram will show no appreciable contribution of a particular type of chromosome combination in micronuclei, since chromosome grouping appears to be a random process, as was shown by the analysis of the number of micronuclei per cell in chapter two, and by cytological data in chapter two and three. Furthermore, the reduction of the number of micronuclei per micronucleated cell, which appears to be the consequence of fusion of micronuclei into a lobed restitution nucleus, gives rise to even more combinations of chromosomes.

    The processes, involved in the formation of micronuclei, are studied in chapter three and four. The effects of the anti-microtubular herbicides APM, oryzalin and the alkaloid colchicine, used for metaphase arrest and induction of micronuclei in mammalian cells, on the mitotic index and micronueleus formation are compared. The disruption of the spindle by direct inhibition of microtubule assemble is responsible for the accumulation of cells at metaphase. The concentrations of the inhibitors required for complete metaphase arrest, vary from 3 μM for APM and oryzalin to 500 μM for colchicine, as a consequence of differences in binding specificity (Hertel et al., 1980; Dustin 1984). The differences in the percentage of ball metaphases indicate specific effects of the above mentioned inhibitors on chromosome scattering. Apart from the disruption of the microtubules, APM and oryzalin have been shown to influence the accumulation of calcium in the mitochondria (Hertel et al., 1981). Moreover, oryzalin disturbs the active excretion of calcium by the plasma membrane. These combined effects result in an increased cytoplasmic calcium concentration (Hertel et al., 1980), which will be higher after oryzalin treatment than after APM treatment, due to the reduction of active calcium excretion by oryzalin. Our 'data suggest that the APM or oryzalin induced increase of the cytoplasmic calcium concentration is involved in both formation and fusion of micronuclei. Colchicine, which does not influence the cytoplasmic calcium concentration, is not effective in the induction of micronuclei. The higher cytoplasmic calcium levels after oryzalin treatment, would increase the fusogenic properties of the nuclear membranes, which would explain why micronuclei exist for a shorter time after oryzalin treatment as compared to APM treatment. This hypothesis will be tested in future experiments by treatments with the calcium ionophore A23187 in combination with the calcium-specific chelator ethyleneglycolbis- (2-aminoethylether)-N,N'-tetra acetic acid (EGTA), with simultaneous measurements of the cytoplasmic calcium concentrations with the new calcium specific fluorochromes Fluo-3 and Rhod-2 (Haugland, 1989).

    In order to obtain both large numbers of micronucleated cells, and large numbers of micronuclei per micronucleated cell, the
    effect of DNA synthesis inhibitors was investigated. The results in chapter five show, that a considerable increase in the number of micronucleated cells can be achieved by HU or APH treatments, and that the time at which micronuclei appear can be controlled. The results further indicate that metaphases have to be exposed to APM for at least 12h, before micronucleation occurs, and that their lifetime is in the same order. These data demonstrate that it is possible to manipulate the conditions of the treatments in order to obtain either a high yield of metaphase chromosomes, or a high yield of micronuclei, with little contamination by micronuclei or chromosomes, respectively. In this way, it becomes possible to determine the moment at which the number of micronuclei per cell is at its maximal value.

    The isolation and characterization of microprotoplasts from micronucleated suspension cells is described in chapter six. Data obtained from DNA content measurements and flow cytometry demonstrate the presence of up to 40% of subprotoplasts with a DNA content less than the G1-level of the APM treated suspension cells. This indicates that genome fractionation has occurred, and the data on the FDA-staining show that most of the subprotoplasts still possess an intact plasma membrane, since FDA can not be retained by vacuolar membranes only (Lesney, 1986). The viability of the microprotoplasts and other types of subprotoplasts is indicated by the successful culture after gradient fractionation. As it is impossible to measure the DNA-content of microprotoplasts in a non-destructive way, no preselection could be performed to use only microprotoplasts for fusion. In a mass fusion system, the smallest microcells will be the least likely to fuse when electrofusion is used, because their small diameter will prevent alignment and membrane breakdown, which are both related to particle diameter (Zimmermann et al., 1982). Individual selection and fusion could overcome this problem (Koop et al., 1983). This control is essential for the efficient application of microprotoplasts, since the DNA content per microprotoplast will depend upon the DNA content per micronucleus in the cell suspension. Microprotoplast fusion will result in transfer of a part of the total number of chromosomes, directly followed by spontaneous chromosome elimination when two distantly related species are fused, since chromosome elimination seems to be directed by genome dose effects (Graves, 1984; Gilissen et al., 1989). Sofar no successful fusion experiments have been performed, which makes it impossible at the moment to comment on the usefulness of microprotoplasts in chromosome transfer. However, fusion experiments with karyoplasts indicate that it is possible to perform fusions in a controlled way (Spangenberg et al., 1987).

    In addition to the microprotoplast fusion, microinjection was developed for transfer of organelles and micronuclei. Glass needles with a large orifice (5pM) were prepared, along with a pressure system, based on the application of mercury. With the injection system, described in chapter seven, it is possible to suck donor material from a donor protoplast, and inject this directly into the recipient. The data on the complementation of the albino tobacco by injection of mature green chloroplasts or etiolated plastids, indicate that protoplasts can survive the injection treatment, and that the injected plastids can be replicated by the recipient. In this way, the organelles to be transferred are not subjected to damaging isolation procedures and they can be preselected visually. Selective transfer of organelles offers a number of advantages when compared to fusion techniques, or transfer of isolated genes. One of the advantages is the protective nature of the membranes associated with chloroplasts, mitochondria and nuclei. Although structural integrity and functionality has been demonstrated for isolated chloroplasts and mitochondria, it is not known whether isolated organelles are still physiologically intact. The isolation of intact nuclei from plant cells has also been described, with data indicating their structural integrity, as well as their ability to transfer genes into recipient protoplasts (Saxena et al., 1986). Transfer of marker genes does not necessarily implicate the functional integrity of isolated nuclei, since transfer of marker genes can be achieved by uptake of isolated genomic DNA. Preliminary results obtained from experiments with microinjection of micronuclei, indicate that it is possible to remove micronuclei from the donor by suction. Sofar, transfer into a recipient has not been achieved. The kanamycine- resistance, which was introduced into N.plumbaginifolia by transformation with Agrobacterium tumefaciens , will be used as selectable marker after transfer of micronuclei. The transfer of chromosomes will be tested with species specific repetitive DNA probes, which are able to discriminate between the donor genome N.plumbaginifolia and the recipient (either Lycopersicon esculentum or Solanum tuberosum ) . Several probes with the required specificity have already been characterized, from a series of highly repetitive sequences, isolated from N.plumbaginifolia (data not shown).

    With the methods, described in this thesis, the transfer of chromosomes via micronuclei has come within reach. Future work will focus on achieving transfer, and study the fate of the introduced micronuclei. This should provide an answer whether micronuclei can be used as chromosome carriers in plants, as has already been shown in mammalian somatic cell genetics.

    Bacterial adhesion
    Loosdrecht, M.C.M. van - \ 1988
    Agricultural University. Promotor(en): A.J.B. Zehnder; J. Lyklema. - S.l. : van Loosdrecht - 113
    micro-organismen - morfologie - cytologie - microbiologie - chemie - colloïden - adsorptie - oppervlakten - oppervlaktechemie - microorganisms - morphology - cytology - microbiology - chemistry - colloids - adsorption - surfaces - surface chemistry

    As mentioned in the introduction of this thesis bacterial adhesion has been studied from a variety of (mostly practice oriented) starting points. This has resulted in a range of widely divergent approaches. In order to elucidate general principles in bacterial adhesion phenomena, we felt it was necessary to start from a fundamental level i.e. using welldefined model systems. In our study colloid chemical principles are applied to microbial systems. Although both colloid chemists and microbiologists have investigated the behaviour of small microscopic particles, there has been only limited cooperation between them in the past. Nevertheless, this study reveals that such a cooperation can be very fruitful.

    After a general (Chapter 1) and a theoretical (Chapter 2) introduction, we deal in Chapters 3 and 4 with the relation between bacterial surface characteristics and adhesion to sulphated polystyrene (a hydrophobic, charged surface). The cell surface hydrophobicity and electrokinetic potential were determined by the contact angle measurement and electrophoresis, respectively. Adhesion increases with increasing bacterial hydrophobicity or decreasing electrokinetic potential. The effect of the electrokinetic potential increases with decreasing hydrophobicity. An interesting finding is the increase with growth rate in surface hydrophobicity of bacteria.

    In Chapter 5 we show that initial adhesion to sulphated polystyrene is reversible and can at least qualitatively be described by the DLVO theory for colloidal stability, i.e., in terms of Van der Waals and electrostatic interactions. From adhesion isotherms we found an adhesion Gibbs energy of -2 to - 3 kT per cell. This corresponds to calculations using DLVO theory that predict adhesion in the so-called secondary minimum, a case where no direct intimate contact is made between bacterium and surface. Finally, the implications of our findings for natural and (bio)technical processes are discussed.

    In Chapter 6 we report on the applicability of the DLVO theory for the interpretation of bacterial adhesion to glass and to more practical surfaces (Rhine river sediment and protein-coated surfaces). In all these cases adhesion could be interpreted in terms of the hydrophobicity and electrical properties of the surfaces.

    The possible influences of adhesion on bacterial activity are discussed in Chapter 7, in the form of a critical literature review. Despite the opinion regularly heard that there might be a direct influence of adhesion on bacterial physiology we have not been able to find any experimental evidence in support of this hypothesis. Different activities of attached and free cells are often due to changes in substrate transport (e.g. diffusion, desorption, or convective transport) or differences in hydrophobicity of active and resting cells. For the conversion of adsorbed substrates the dissolved concentration determines the conversion rate. With strongly adsorbing compounds the conversion can become desorption-limited, whereas non-desorbing compounds are often not degraded.

    In this thesis it is shown that application of colloid chemistry to microbial systems can lead to interesting new viewpoints. More specifically, the DLVO theory for colloidal stability was found to give a quantitative description of the initial stage of bacterial adhesion both to model surfaces as in more applied situations (Chapters 5 and 6). Generally, in the studies dealing with interaction between bacteria themselves or between bacteria and surfaces electrostatic interactions are often neglected, despite the fact that this interaction is often desicive whether strong adhesion can occur or not.

    The insights derived from a colloid chemical approach can be used, as complementary to a more biological approach, in understanding the (auto-) immobilization of bacteria in natural and biotechnological systems, as e.g. in UASB- reactors.

    The experimental methods developed in this study may also be successfully applicable in other research areas. Due to the sensitivity of the contact angle and electrophoretic mobility measurements they can for instance be applied as a rapid screening
    method of new isolates or cell surface mutants. Especially with urface mutants the methods mentioned here are much faster than conventional biochemical or immunological methods.

    The contact angle and electrophoretic mobility measurements may also be useful for obtaining information on the structure of the outer part of the cell wall. In particular electrophoresis, at different pH and electrolyte strength, combined with chemical modifications of specific groups (e.g. -NH 2 groups) may be very powerful. Preliminary experiments with lipopolysaccharide mutants of Pseudomonads are very promising. For this and other applications it is necessary to improve the electrochemical characterization of bacteria, especially with respect to the influence of bacterial conductivity.

    Other areas in microbiology that may be successfully treated by colloid chemical theories concern firstly the biological availability of substances, in particular micro-pollutants, to bacteria. This availability is mainly determined by substrate adsorption to inert solid material and substrate transport through the cell wall and membrane. A second interesting field might be the relation between molecular composition and function or stability of membranes in different bacteria, or under different environmental conditions.

    The function of vesicles in the actinomycete Frankia
    Meesters, T. - \ 1988
    Agricultural University. Promotor(en): A.J.B. Zehnder, co-promotor(en): A.D.L. Akkermans. - S.l. : Meesters - 114
    cytologie - ontwikkeling - frankia - groei - micro-organismen - morfologie - cytology - development - frankia - growth - microorganisms - morphology

    The actinomycete Frankia is a symbiotic nitrogen fixer, living in root nodules of many non-leguminous plants. A typical characteristic of this endophytic organism is the formation of specialized swollen cell structures, called vesicles. Frankia vesicles have been brought In relation to nitrogen fixing activity, but many questions about their formation and their function still had to be answered. The present study was done to investigate the function of these vesicles. First, an electronmicros copy study has been initiated to investigate the ultrastructure of Frankia cells. The vesicle envelope was studied in detail, and shown to be a multilaminate structure, which was in accordance with other published results. The vesicle envelope was continuous with the hyphae cell wall. It was also shown that no difference was found in the ultrastructure of vesicles with, or without induced nitrogenase (chapter II). The formation of vesicles in different Frankia strains was investigated by comparing a strain with, and one without vesicle formation in the presence of ammonia. In the presence of ammonia, nitrogenase is generally repressed. The vesicles that were formed in the medium with ammonia did not contain nitrogenase (chapter III).

    Frankia nitrogenase could be detected by using antisera against nitrogenase from Rhizobium leguminosarum or from Azotobacter vinelandii. The latter appeared to be very specific, and was used for localization of nitrogenase on ultrathin cryosections. It could be shown that nitrogenase is present uniquely in the vesicles in Frankia strains Ccl.17 and EAN1pec (chapters IV-1, IV-2). In these cells, probably a reducing environment is maintained, thus preventing inhibition of nitrogenase by oxygen. In accordance herewith, it has been reported that one Frankia strain (Cc13) was able to fix nitrogen without forming vesicles, under microaerobic conditions. The Influence of microaerobic conditions on the localization of nitrogenase in strain Ccl. 17 was tested. In this strain, no change of the localization of nitrogenase in the vesicles with reduced oxygen partial pressure has been observed (chapter IV-3). From these results, it can be concluded that nitrogenase is generally located within the vesicles, although vesicle formation and nitrogenase induction can be controlled independently in some strains. The mechanism of regulation of the strict localization is still unknown. One of the possibilities of repression of nif- genes is the occurrence of DNA rearrangement during vesicle differentiation. This possibility has been investigated by comparing DNA from vesicle clusters from nitrogen fixing root nodules with DNA from hyphae from the corresponding pure culture. No differences could be detected in the nif -genes from these two preparations. The results mean that the question, how nif- gene expression i s repressed in the hyphae of Frankia, still need to be answered. The progress made in the past years in Frankia molecular genetics promises that many still open questions about regulation mechanisms in Frankia will be answered in the near future.

    Localization of viral antigens in leaf protoplasts and plants by immunogold labelling
    Lent, J.W.M. van - \ 1988
    Agricultural University. Promotor(en): J.P.H. van der Want; B.J.M. Verduin. - S.l. : van Lent - 125
    bromovirus - cellen - vignabonen - cytologie - histologie - methodologie - plantenziekten - plantenvirussen - relaties - technieken - vigna - virologie - virussen - bromovirus - cells - cowpeas - cytology - histology - methodology - plant diseases - plant viruses - relationships - techniques - vigna - virology - viruses

    This thesis describes the application of an immunocytochemical technique, immunogold labelling, new in the light and electron microscopic study of the plant viral infection. In Chapter 1 the present state of knowledge of the plant viral infection process, as revealed by insitu studies of infected cells, is briefly reviewed. Until now, light and electron microscopic studies have merely described morphological changes in cells and tissue as a result of viral infection, but have failed to provide information on the functional role of these structures in the viral infection process and their association with viral components. A common cytopathological feature of many different plant viruses seems to be the induction of membranous vesicles or membranous bodies, which have been implicated in viral replication. However, only in a few cases some evidence was obtained with regard to the Intracellular location of viral replication and the association of replication and membranes. Available cytochemical techniques have apparently failed to provide a tool for the identification of virus particles and virus-encoded proteins within cellular structures. The Impact of a suitable detection techniques to elucidate the molecular processes of viral replication and transport insitu is obvious, as it would link findings obtained by invitro experiments to the events observed in the cell.

    Immunogold labelling seems to provide such a tool for the tracing of antigens in light and electron microscopic preparations of biological specimens. Gold particles are excellent markers for electron microscopy, because of their high electron density which makes them appear as black dots In EM preparations. Furthermore, by a simple silver staining following gold labelling, viral antigens can be dete cted in semi-thin sections with the light microscope. The application of immunogold labelling for the light and electron microscopic localization of antigens is described in Chapters 2, 3, 4, 5, 6 and 7.

    In Chapter 2 the preparation of homodisperse suspensions of colloidal gold particles is described. By adsorption of protein A to the surface of the gold particles, a marker (protein A-gold, pAg) is obtained which can be used for labelling antigen-antibody complexes. The specificity of the technique was demonstrated by gold labelling of antibodies bound to plant viruses in mixed suspensions of two viruses. Each virus was labelled using its homologous antiserum and pAg, and no significant cross-reaction with the other virus occurred. Simultaneous identification of two different viruses (CCMV and SBMV) with similar morphological appearance was achieved by double labelling with pAg-complexes containing gold particles of 7 and 16 nm, respectively. Immunogold labelling of viral antigens in suspension has been applied to distinguish between different serologically related viruses like strains of TMV (Pares and Whitecross, 1982), and the potyvirus sugarcane mosaic virus and maize dwarf mosaic virus (Alexander and Toler, 1986; 1985). A clear advantage of the immunogold labelling over conventional decoration of antigens is that the discrete gold particles allow quantification of the results.

    The immunogold labelling of viral antigen in ultrathin sections of infected protoplasts is described in Chapter 3. Best results were obtained when the protoplasts were only mildly fixed with aldehydes, dehydrated and finally embedded in Lowicryl K4M at -30°C. The antigenicity of viral coat protein was well preserved. A disadvantage of the method is the limited preservation of cell structures, especially membranes due to extraction of lipids. Weibull etal. (1983) reported that approximately 50% of the lipid content of cells may be extracted, despite the low temperatures used in the Lowicryl K4M embedding procedure. Ashford etal. (1986) questioned the low temperature character of Lowicryl embedding, and found that during polymerization of the resin, temperature rises due to the exothermic nature of the reaction. With plant tissue (not protoplasts), low temperature dehydration and infiltration of the embedding resin must be prolonged, to allow sufficient penetration of the chemicals through the thick walls surrounding the plant cells, and this may result in even more extraction than reported by Weibull and colleagues. Rapid dehydration in ethanol and infiltration of plant tissue with a polar resin like LR White at ambient temperatures, therefore, seems to be a good alternative (Newman etal. , 1983; Causton, 1984; Newman and Jasani, 1984).

    Light microscopic localization of viral antigen in semi-thin sections of LR White embedded plant tissue is described in Chapter 6. CCMV was successfully localized in petiolules of systemically inoculated cowpea plants by immunogold labelling and subsequent silver staining (immunogold/silver staining: IGSS). The silver stain could be observed in the light microscope by brightfield, darkfield and phase-contrast illumination. Most sensitive detection, however, was obtained with epi- illumination using polarized light (epipolarization microscopy). Combining epipolarization illumination with brightfield illumination allowed the simultaneous observation of silver stain and cell morphology.

    Immunogold labelling and IGSS in combination with appropriate fixation and embedding of biological specimens, appear to be efficient and simple techniques for the insitu identification and localization of antigens, with many advantages over other immunochemical and cytochemical techniques, like ferritin- labelling, peroxidase-anti-peroxidase, immunofluorescence and autoradiography, which have only incidentally been used in plant virus research. Recently, Patterson and Verduin (1987) have reviewed the literature on the use of immunogold labelling in animal and plant virology, showing numerous fields of applications and discussing progress made in virus research. With respect to the technique the authors rightly concluded that immunogold labelling is a flexible technique with little limitation for the improvement of existing assays and the development of new ones.

    Using immunogold labelling to identify and localize virus particles and coat protein, CCMV- infection in cowpea protoplasts was studied as function of the infection time. Observations with regard to virus entry into protoplasts are reported in Chapter 3. Upon inoculation aggregates of virus particles were observed attached to the plasmamembrane, or sometimes penetrating the plasmamembrane at places where the membrane appeared to be damaged. Virus was also found inside vesicles formed by invagination of the plasmamembrane. These vesicles with inoculum-virus particles were stable over long periods of time. Large vesicles (vacuoles) containing viral antigen were also detected at 24 h post-inoculation in protoplasts which were not infected by CCMV.

    The mechanism by which plant viruses enter their host cells is still disputed (Shaw, 1986). Passage of the plasmalemma by endocytosis was suggested by Takebe (1975), and through pores or lesions by Burgess etal. (1973) and Watts etal. (1981). Our observations do not favour endocytosis to be the mechanism of virus entry leading to infection of the protoplasts as virus containing vesicles are stable. Recently, Roenhorst etal. (1988) presented data supporting a mechanism of virus entry by initial physical association of virus particles with the protoplast membrane and subsequent invasion of virus particles through membrane lesions. Such a mechanism may be also applicable to the cytoplasmic extrusions observed by Laidlaw (1987) after puncturing plant epidermal cells. The author suggested that virus particles may adsorb to the plasmalemma covering the extrusions, which are then withdrawn into the cell. Invasion of whole particles through membrane lesions may then be followed by a uncoating and initial translation (cotranslational disassembly) at the cytoplasmic ribosomes as suggested by Wilson (1985).

    Ultrastructure of RNA-inoculated protoplasts was studied in sections of aldehyde- and osmium-fixed protoplasts (Chapter 4). Cytological alterations attributed to virus infection consisted of dilation of the endoplasmic reticulum (ER) and the formation of vesicles early in infection. Distended ER and vesicles seemed to form a kind of membranous area in the cytoplasm. In protoplasts fixed and embedded in Lowicryl K4M newly synthesized virus particles or coat protein were first localized in restricted areas of the cytoplasm at 6-9 h post-inoculation. The rough appearance of the cytoplasm in these areas suggested the presence of membranous structures like observed in osmium-fixed protoplasts. However, due to poor membrane preservation in Lowicryl embedded material this could not be proven. Within one protoplast several of these labelled areas were identified. At later stages of infection viral antigen was located throughout the cytoplasm, but also in the nucleus and in particular the nucleolus. No viral antigen was detected in or specifically associated with chloroplasts, mitochondria, microbodies and vacuoles. The specificity of gold labelling was demonstrated by quantification of the labelling density on sections of infected and non-infected protoplasts. These results indicate that CCMV coat protein synthesis and virus assembly take place in the cytoplasm of plant cells, but the involvement of cellular structures, in particular membranes, remains to be established. Protein synthesis and virus assembly may occur in certain restricted sites (compartments) in the cytoplasm possibly formed by the membranous bodies. Compartmentalization of the cytoplasm, creating different environments in the cell, may explain the occurrence of both disassembly and assembly in the same cell, and furthermore account for the phenomenon of specific assembly of viral RUA and homologous coat protein in cells infected with two related viruses like CCMV and BMV (Sakai etal. , 1983 ; Zaitlin and Hull, 1987). Whether RNA-replication also occurs in the same location as coat protein synthesis and virus assembly could be established by localization of non-structural virus encoded proteins involved in viral replication. However, antisera against these products of the CCMV-genome were not available. The function of CCMV coat protein or virus in the nucleus and especially the nucleolus is not known. Coat protein may have an affinity for ribosomal proteins and/or fulfill some functional role in the viral replication. Kim 1977 described the occurrence of filamentous inclusions (FI) in the nucleus often associated with the nucleolus. These FI were not found in the nuclei of cowpea protoplasts (this study) or tobacco protoplasts (Burgess etal. , 1974), but may be formed later in the infection by excess coat protein. Bancroft etal. (1969) showed the ability of CCMV-coat protein to form narrow tubules under specific conditions. The (FI) described by Kim (1977) may represent this type of coat protein aggregation, although the chemical composition of the (FI) is not yet known.

    In Chapter 5 preliminary observations are reported on the localization of sites of CPMV replication in cowpea protoplasts, by in situ detection of coat proteins and non-structural proteins involved in viral replication and proteolytic processing. With regard to virus entry and subsequent locations of inoculum virus inside vesicles, similar phenomena were observed as in infection with CCMV. Infection of CPMV generates large inclusion bodies in the cytoplasm, consisting of membranous vesicles with fibrillary material and adjoining amorphous electron-dense material which have been observed as early as 12 h post- inoculation. Virus particles and/or coat protein were first detected 24 h after inoculation throughout the entire cytoplasm and in between the membranous vesicles and electron dense material. The 24K, 170K and their precursor proteins were exclusively localized in the electron dense material and not in association with the membranous vesicles or any other location in the cell. These results show that the electron-dense material consists at least in part of CPMV-encoded non-structural proteins and may represent a site for accumulation of non-functional proteins. The membranous vesicles have been implicated in viral RNA synthesis (Goldbach and Van Kammen, 1985). The failure to detect non- structural proteins in association with these membranes may be explained by either a low concentration of these proteins at the site of replication or by extraction of these proteins during the fixation and embedding procedure, despite the low temperature.

    With IGSS the distribution of CCMV in cowpea plants was monitored at different times after systemic inoculation according to Dawson and Sehlegel (1976) (Chapters 6 and 7). No virus was detected at the time of temperature shift (t=0) in petiolule and leaves of plants subjected to 3 days of differential temperature treatment. Virus was first localized in phloem parenchyma cells of petiolule and veins at t=3 h and from there it spread to neighbouring tissues. Twenty four hours after systemic inoculation virus was located in the phloem, bundle sheath, cortex, but also in the cambium and some xylem cells. These results show that CCMV is transported from the inoculated primary leaves to the secondary leaves through the phloem, apparently following the route of metabolites. This finding is in agreement and further supports the generally accepted concept of plant virus long-distance transport through phloem. tissue (Matthews, 1982; Atabekov and Dorokhov, 1984). The failure to detect CCMV in differentiated sieve elements may indicate that the form in which the infectious entity is transported is another than virus particles (Atabekov and Dorokhov, 1984), or that the amount of virus transported through the sieve elements is below detectable levels. The true character of the synchrony of infection of leaf mesophyll cells obtained by differential temperature treatment is disputed. Infection of mesophyll tells may have been accomplished after shifting the plants to higher temperature by fast transport of infectious particles from the vascular tissue, as was also suggested by Dorokhov etal. (1981).

    For the first time a suitable method for localization of antigens is available, which can be routinely applied for both light and electron microscopic study of the plant viral infection process. The application of the gold labelling technique in the localization of viral structural and non-structural proteins has been demonstrated, using CCMV- and CPMV-infections of plant cells as model system.

    With regard to the technique, future work must be done on the improvement of the preservation of cellular structures, especially membranes, as these appear only poorly in Lowicryl embedded plant tissue even with dehydration, infiltration and polymerization at low temperatures. Alternatives, may be found in cryofixation and cryosectioning or freeze-substitution techniques.

    With regard to the study of the plant viral infection process, the localization of virus-encoded proteins involved in replication and transport, but also the localization of plant viral nucleic acids by insitu hybridization, will contribute to the understanding of the mechanisms underlying these events. New biochemical techniques like the production of infectious transcripts from cloned viral cDNA (Ahlquist etal. 1984) enabling genetic manipulation of the viral genome, and integration of plant viral genes into the plant genome (Gardner etal. , 1984; Abel etal. , 1986) will supply future model systems for the study of virus-host interactions.

    Plant cells : immobilization and oxygen transfer
    Hulst, A.C. - \ 1987
    Agricultural University. Promotor(en): J. Tramper, co-promotor(en): K. van 't Riet. - S.l. : Hulst - 121
    plantkunde - cellen - cytologie - immobilisatie - metabolisme - voeding - plantenfysiologie - celmetabolisme - celfysiologie - geïmmobiliseerde cellen - botany - cells - cytology - immobilization - metabolism - nutrition - plant physiology - cell metabolism - cell physiology - immobilized cells

    The study described in this thesis is part of the integrated project 'Biotechnological production of non-persistent bioinsecticides by means of plant cells invitro ' and was done in close cooperation with the research Institute Ital within the framework of NOVAPLANT. The plant cells used in this project were Tagetes species which produce thiophenes, naturally occurring biocides, particularly against nematodes.

    The objective of the study described in this thesis was to use immobilized plant cells or large plant cell aggregates for secondary metabolite production. In particular the upscaling of immobilization techniques for plant cells, the role of diffusion limitation of oxygen as a substrate on the immobilized plant cells and its effect on secondary metabolite production of the immobilized plant cells were subject of research.

    A literature survey on immobilized plant cells is presented in Chapter 2. The advantages of immobilized plant cells, several aspects concerning immobilization techniques, consequences of plant cell immobilization, immobilized plant cell reactors, and future prospects of immobilized plant cells are discussed in this Chapter.

    Chapter 3 deals with the application of the resonance nozzle as an immobilization technique with a high production capacity for plant cells as well as yeast cells in calcium alginate gel beads. It is found that this technique has a production capacity of two orders of magnitude larger than the conventional dripping technique with a needle. The viability of the cells after immobilization with the resonance nozzle was preserved. An extension of the applicability of the nozzle technique for thermogelling gel supports (k-carrageenan, agar and gellan gum) Is described in Chapter 4. Plant cells, yeast cells, bacterial cells and insect cells were used as model systems in the experiments.

    In Chapter 5, the occurrence of oxygen diffusion limitation of Daucuscarota cells in agarose, calcium alginate and (κ-carrageenan, is determined by respiration measurements of the immobilized cells in order to explain the enhanced pronounced secondary metabolite production with alginate immobilized plant cells from in the literature reported experiments. However, in our experiments no differences between the support materials could be observed.

    The effective diffusion coefficient for oxygen (ID e ) in the gel material is an important factor in mathematical model calculations in order to quantify the occurrence of oxygen diffusion limitation. Chapter 6 deals with the experiments in which (ID e ) was determined in different gel materials (calcium alginate, κ-carrageenan, gellan gum, agar and agarose) by measuring the oxygen diffusion from a well- stirred solution into gel beads, which were initially free of oxygen. A mathematical model was fitted on the experimental data resulting in the value of (ID e ) which was used In the following experiments.

    In several parts of this thesis a mathematical model was used for calculation of oxygen concentration profiles in gel beads containing plant cells or cell aggregates in order to visualize the occurrence of oxygen diffusion limitation. In Chapter 7 this model is tested on validity by experimental measurement of the oxygen concentration profiles in agarose beads containing respiring plant cells of Tagetesminuta . This was done with the aid of an oxygen microelectrode with a tip of 15 μm. The experimental and calculated oxygen concentration profiles correspond quite well.

    Chapter 8 deals with the effects of aggregate size and oxygen diffusion limitation on thiophene production and cell growth by cell aggregates of Tagetespatula . It is concluded that aggregate size is related to thiophene production by the observation of an 'optimum' aggregate size where the production is highest. Calculations of the oxygen concentration profiles showed that this could be due to the absence of oxygen In the centre of the aggregates.

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