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Staff Publications

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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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Identification of novel auxin responses during Arabidopsis embryogenesis
    Rademacher, E.H. - \ 2009
    Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Dolf Weijers. - [S.l. : S.n. - ISBN 9789085854906 - 110
    arabidopsis - embryogenese - plantenfysiologie - auxinen - celfysiologie - arabidopsis - embryogenesis - plant physiology - auxins - cell physiology
    Plants normally form one embryo per seed. Under special circumstances, such as death of the embryo, a second embryo can develop from a supportive structure called the suspensor. These suspensor cells therefore provide a reservoir of stem cells for the generation of secondary embryos. At the start of this project, the mechanisms that control the formation of secondary embryos were completely unclear.
    By conducting a systematic screen for cellular responses to the plant hormone auxin during embryogenesis we found that auxin prevents embryo development from suspensor cells. The detailed analysis of auxin response components allowed us to identify the auxin-dependent transcription factors that mediate auxin action in the suspensor. Furthermore, we found that the control of expression of these auxin response transcription factors contributes to early embryo pattern formation. This work identified the first molecular players in the control of suspensor-embryo transformation and provides a stepping stone for elucidating the genetic networks that control embryo identity in plants.

    The role of macromolecular stability in desiccation tolerance
    Wolkers, W.F. - \ 1998
    Agricultural University. Promotor(en): L.H.W. van der Plas; F.A. Hoekstra. - S.l. : Wolkers - ISBN 9789054858805 - 244
    vloeistoffen (liquids) - absorptie - emissie - omloop - veroudering - abcissie - degeneratie - necrose - verouderen - verwelking - biochemie - metabolisme - polymeren - moleculaire biologie - biofysica - eiwitten - enzymen - nucleïnezuren - celfysiologie - liquids - absorption - emission - circulation - senescence - abscission - degeneration - necrosis - aging - wilting - biochemistry - metabolism - polymers - molecular biology - biophysics - proteins - enzymes - nucleic acids - cell physiology

    The work presented in this thesis concerns a study on the molecular interactions that play a role in the macromolecular stability of desiccation-tolerant higher plant organs. Fourier transform infrared microspectroscopy was used as the main experimental technique to assess macromolecular structures within their native environment.

    Protein secondary structure and membrane phase behavior of Typha latifolia pollen were studied in the course of accelerated aging. The overall protein secondary structure of fresh pollen highly resembled that of aged pollen, which indicates that endogenous proteins in these pollen are very stable, at least with respect to their conformation. In contrast, large changes in membrane phase behavior were detected between fresh and aged pollen. Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membranes of aged pollen were at least partly in the gel phase (Chapter 2).

    The in situ heat stability of the proteins in this pollen was studied as a function of the water content of the pollen. Temperature-induced denaturation of proteins was accompanied by the formation of intermolecular extendedbeta-sheet structures. Below 0.16 g H 2 O g -1dry weight (DW), the temperature at which the proteins began to denature increased rapidly and the extent of protein structural rearrangements due to heating decreased (Chapter 3).

    Inspection of the overall protein secondary structure of thin slices of embryo axes of onion, white cabbage and radish seeds did not show signs of protein aggregation and denaturation after long-term dry storage. It was concluded that, despite the loss of viability and the long postmortem storage period, secondary structure of proteins in desiccation-tolerant dry seed is very stable and conserved during at least several decades of open storage (Chapter 4).

    Adaptations in overall protein secondary structure in association with the acquisition of desiccation tolerance were studied using isolated immature maize embryos. Isolated immature maize ( Zea mays ) embryos acquire tolerance to rapid drying between 22 and 25 days after pollination (DAP) and to slow drying from 18-DAP onwards. In fresh, viable 20- and 25-DAP embryo axes, the overall protein secondary structure was identical, and this was maintained after flash drying. On rapid drying, 20-DAP axes showed signs of protein breakdown and lost viability. Rapidly dried 25-DAP embryos germinated and had a protein profile similar to the fresh control. On slow drying, thealpha-helical contribution in both the 20- and 25-DAP embryo axes increased when compared with that in the fresh controls, and survival of desiccation was high. The protein profile in dry mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in 20-DAP embryo axes and much less so in 25-DAP axes. After slow drying, membrane integrity was retained in both the 20- and 25-DAP axes. It was concluded that slow drying of excised immature embryos leads to an increased proportion ofalpha-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress (Chapter 5).

    A novel FTIR method was used to study glasses of pure carbohydrates and glasses in the cytoplasm of desiccation-tolerant plant organs. The method is based on a temperature study of the position of the OH-stretching vibration band (vOH). The glass transition temperatures ( Tg s) of several dry carbohydrate glasses determined by this FTIR method resembled those of produced by other methods. FTIR analysis gives additional information on the molecular properties of glassy structures. The shift ofvOH with temperature - the wavenumber-temperature coefficient (WTC) - is indicative of the average strength of hydrogen bonding in glasses. The WTC was found to be higher in sugar glasses having higher Tg . This suggests that carbohydrate glasses are more loosely packed when they have higher Tg . For Typha latifolia pollen and dried Craterostigma plantagineum leaves similarvOH vs temperature plots were obtained as for pure carbohydrate glasses, indicating that a glass transition was observed. The data suggested that the carbohydrates that are present in the cytoplasm of these plant organs are the primary components contributing to the glassy state (Chapter 6).

    In order to find a relation between desiccation tolerance and physical stability, the heat stability of proteins and the properties of the glassy matrix in several dry maturation-defective mutant seeds of Arabidopsis thaliana were studied. Proteins in dried wild-type seeds did not denature up to 150°C. In dried desiccation-sensitive lec1-1 , lec1-3 and abi3-5 seeds, protein denaturation occurs at temperatures below 100°C. In desiccation-tolerant abi3-7 and abi3-1 seeds, protein denaturation commenced above 120 and 135°C, respectively. The maximal rate of change ofvH with temperature was much higher in abi3-5 , lec1-1 and lec1-3 mutant seeds than in wild-type, abi3-1 , and abi3-7 seeds. This was interpreted as a higher molecular packing density in dried desiccation-tolerant than in dried desiccation-sensitive seeds, which is associated with a higher, respectively lower protein denaturation temperature. The generally lower physical stability of the desiccation-sensitive mutant seeds coincides with a lack of biochemical adaptations that normally occur in the later stages of seed development (Chapter 7).

    The relation between physical stability and desiccation tolerance was also studied in slowly dried (desiccation-tolerant) and rapidly dried (desiccation-sensitive) carrot somatic embryos. Although protein denaturation temperatures were similar in the embryos after slow or rapid drying, the extent of protein denaturation was higher in the rapidly dried embryos. Slowly dried embryos are in a glassy state at room temperature, whereas no clearly defined glass transition temperature was observed in the rapidly dried embryos. Moreover, the molecular packing density of the cytoplasmic glassy matrix was higher in the slowly dried embryos. While sucrose is the major soluble carbohydrate after rapid drying, on slow drying, the trisaccharide umbelliferose accumulates at the expense of sucrose. Dry umbelliferose and sucrose glasses have almost similar Tg s. Both umbelliferose and sucrose depressed the transition temperature of dry liposomal membranes equally well; prevented leakage from dry liposomes after rehydration, and preserved the secondary structure of dried proteins. The similar protecting properties in model systems and the apparent interchangeability of both sugars in viable dry somatic embryos suggest no special role for umbelliferose in the improved physical stability of the slowly dried somatic embryos. It was suggested that LEA proteins, which are synthesized during slow drying together with the sugars, are responsible for the increased stability of the slowly dried embryos (Chapter 8).

    The dehydration-sensitive polypeptide, poly-L-lysine was used as a model to study dehydration-induced conformational transitions of this polypeptide as influenced by drying rate and carbohydrates. In solution poly-L-lysine adopts a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, the polypeptide adopts an extendedbeta-sheet conformation. Upon fast air-drying within 2-3 minutes, the aqueous polypeptide structure is preserved. Slow air-drying in the presence of sugars also preserves the aqueous conformation and results in the formation of a glassy state having a higher Tg than that of sugar alone. The importance of direct sugar - polypeptide interaction in stabilization during slow air-drying was studied by drying the polypeptide in the presence of glucose, sucrose or dextran. Compared to dextran (and sucrose to a lesser extent), glucose gives superior protection, while having the lowest Tg and the best interacting properties. It was suggested that during slow drying, a protectant with sufficient interaction is required for preservation of the aqueous protein structure (Chapter 9).

    The structure of a D-7 LEA (late embryogenesis abundant)-like protein protein isolated from Typha latifolia pollen was studied using FTIR. In solution, the protein adopts a random coil conformation. Fast air-drying (5 minutes) leads to the formation ofalpha-helical structure, whereas slow drying (few hours) leads to bothalpha-helical and intermolecular extendedbeta-sheet structures. When dried in the presence of sucrose, the protein adopts predominantlyalpha-helical conformation, irrespective of drying rate. Drying of a mixture of LEA protein and sucrose results in the formation of a glassy state having higher Tg and a higher average strength of hydrogen bonding than a pure sucrose glass. It was suggested that LEA proteins might be involved in the formation of a tight molecular network in the dehydrating cytoplasm of anhydrobiotic organisms, which may contribute to desiccation tolerance (Chapter 10).

    Taken together, in situ FTIR studies can give additional information on the molecular organization in desiccation-tolerant cells. The added value of this approach is that molecular structures and inter-molecular interactions can be studied in intact biological systems (Chapter 11).

    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.

    The Tapetum: cytology, function, biochemistry and evolution
    Hesse, M. ; Pacini, E. ; Willemse, M.T.M. - \ 1993
    Berlin : Springer Verlag - ISBN 9783211824863 - 152
    biochemie - embryozak - metabolisme - stuifmeel - polymeren - sporen - celfysiologie - biochemistry - embryo sac - metabolism - pollen - polymers - spores - cell physiology
    The anther tapetum, present in all land plants, is a highly specialized, transient tissue surrounding the (micro-)spores and/or pollen grains during their development.Any tapetum malfunction causes male sterility. The exact knowledge of tapetum form and function therefore is indispensable not only for basic research, but also and especially in plant breeding and plant genetics.In fourteen contributions by reknown experts, a comprehensive account of the various characters and functions of the tapetum is provided, covering the areas of cytology, cytophysiology, biochemistry, tapetum development and function.
    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.

    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.

    Regulation of the development of the first leaf of oats (Avena sativa L.) : characterization and subcellular localization of proteases
    Valk, H.C.P.M. van der - \ 1987
    Agricultural University. Promotor(en): J. Bruinsma; L.C. van Loon. - S.l. : Van der Valk - 111
    avena sativa - cytologie - glycosyltransferasen - hexosyltransferases - bladeren - haver - papaïne - pentosyltransferasen - pepsine - fosforylase - plantenontwikkeling - plantenfysiologie - proteïnasen - trypsine - celfysiologie - avena sativa - cytology - glycosyltransferases - hexosyltransferases - leaves - oats - papain - pentosyltransferases - pepsin - phosphorylase - plant development - plant physiology - proteinases - trypsin - cell physiology

    The loss of chlorophyll during the senescence of leaves is preceded by a decrease in protein content. Proteases responsible for the degradation of the proteins have been implicated in the regulation of the senescence process. The first leaf of the seedling of oats ( Avena sativa L.) demonstrates the typical pattern of leaf senescence in cereals and was chosen to study the properties and subcellular localization of proteases throughout leaf development.

    In Chapter 1, a general introduction is given that indicates the significance of the breakdown of proteins in leaves for seed yield and quality in cereals. Also in this chapter, protease classification is documented, and the possible role of changing protease activities in the regulation of protein degradation is discussed.

    Oat leaves contain two major proteases with pH optima at pH 4.5 ("acidic protease") and 7.5 ("neutral protease"). During natural development of greenhouse-grown plants both types of enzymes showed highest activities in young and fully-grown leaves, and decreased throughout the course of senescence. Also in detached leaves incubated in the dark and, therefore, subject to accelerated ageing, loss of protein was not accompanied by increases in protease activities. In the light, protease activities increased, but the rate of protein loss was greatly reduced. Thus, protein breakdown appears to be independent of the amount of protease present and additional synthesis of the major proteases is not required for protein loss during senescence (Chapter 1).

    This apparent paradox could be resolved if proteases and their substrates were spatially separated and brought into contact by a controlled decompartmentalization. Therefore, the distribution and subcellular localization of the major proteases were determined. Protoplasts were prepared as a first step in the isolation of vacuoles. However, the cell wall-degrading enzyme mixtures used for protoplast isolation were seriously contaminated by proteases interfering with the determination of the endogenous protease activity in the isolated protoplasts. These extra proteases could be inactivated by heating the cell well-degrading enzyme mixture at 50°C for 10 min at pH 6.5. This treatment did not impair the cell walldegrading activity. Protoplasts isolated with heated enzymes showed similar protease activities as washed protoplasts, isolated with untreated enzymes. This proved that contaminating proteases were effectively removed during protoplast washing, and that the protease activity measured in isolated protoplasts was derived from the protoplasts themselves. (Chapter 3).

    Vacuoles were isolated by osmotically lysing the protoplasts in the presence of K 2 HPO 4 . The maximum concentration of phosphate by which lysis occurred decreased progressively with increasing leaf age. By using the appropriate phosphate concentrations, it became possible to isolate clean vacuoles from leaves up to an advanced stage of senescence, when the leaves had lost more than 50% of their protein. From leaves older than 17 days, only vacuoplasts (vacuoles with adhering cytoplasm, within a resealed plasma membrane) could be obtained. The integrity of both the plasmalemma and the tonoplast decreased in these older leaves and this phenomenon might be linked with increased decompartmentalization at a late stage of senescence (Chapter 4).

    When the distribution of the proteases was determined in different subcellular fractions, on an average 16% of the acidic protease activity was washed out of the intercellular space of the leaves. The major part of the acidic activity was located within the vacuole. The neutral protease was absent from both these compartments and must, therefore, be cytoplasmic. During the course of leaf development, all of the acidic protease activity present in protoplasts was recovered in the vacuoles, as long as clean vacuoles could be isolated (i.e. up to 17 days). It seems most likely that protein degradation is controlled by import of protein substrates into the vacuole (Chapter 5).

    The acidic and neutral proteases were partly purified by gel filtration and anion-exchange chromatography (Chapter 6). The enzymes hardly separated, indicating that they have similar molecular weights and charges. The neutral activity was stabilized by merceptoethanol and inhibited by inhibitors of metallopeptidases, whereas the acidic one was not. Both activities were inhibited to varying extents by sulphydryl- and serinetype inhibitors. Both enzymes were endopeptideses. The instability of, in particular, the neutral protease seriously hampers its further purification and characterization.

    Furthermore, by activity staining after electrophoretic separation and using aminoacyl-2-naphthyl am ides as substrates, five aminopeptidases and one trypsin-like endopeptidase were identified (Chapter 7). The main aminopeptidase was largely unspecific. Two aminopeptidases showed preference for arginine and lysine. An iminopeptidase acted on proline. The fifth aminopeptidase was most active with methionine. All enzymes were active at pH 4.5, 6.0 and 7.5, but showed highest activities at low rather then at neutral pH. The trypsine-like endopeptidase was active at pH 4.5 and 7.5, was inhibited by o -phenanthroline and was not identical with either the acidic or the neutral protease described previously. Its activity decreased during leaf development. During storage of protein extracts in the cold this arginine-specific endopeptidase associated with ribulosebisphophate carboxylase, concomitant with a loss of this protein band. As to how far this enzyme has a function in protein breakdown invivo has to be further elucidated.

    The results of this study show that the levels of protease activities are not correlated with the rate of over all protein degradation. The acidic protease has no entry to the substrates to be degraded. In contrast, the neutral protease appears to be located together with protein substrates and organelles in the cytoplasm, but seems to selectively degrade only a few proteins. Whereas the mechanism of normal protein turnover remains to be elucidated, the slow, steady decline of protein during natural development may be controlled by, on the one hand, compartmentalization of the acidic protease in the vacuole and, on the other hand, by maintaining a pH removed from the optimum for exopeptidese action in the cytoplasm.

    Isolatie en analyse van plantaardige celwanden
    Pannebakker, E.G. - \ 1983
    Wageningen : CABO (CABO-verslag no. 46) - 30
    celwanden - cytologie - histologie - plantenfysiologie - celfysiologie - cell walls - cytology - histology - plant physiology - cell physiology
    Celbiologie : een tweeluik
    Jongkind, J.F. - \ 1978
    Wageningen : Landbouwhogeschool - 26
    biofysica - celfysiologie - biophysics - cell physiology
    Insluitingen van microorganismen in schimmels
    Anonymous, - \ 1976
    Wageningen : [s.n.] (Literatuurlijst / Centrum voor landbouwpublikaties en landbouwdocumentatie no. 3915)
    bibliografieën - cytologie - schimmels - mycologie - plantenfysiologie - celfysiologie - bibliographies - cytology - fungi - mycology - plant physiology - cell physiology
    The potential response of plasmalemma tonoplast and cell wall upon photosynthetic energy conversion in Nitella
    Vredenberg, W.J. - \ 1971
    Wien : [s.n.] (Publication / Plantphysiological research centre no. 110) - 6
    plasmamembranen - celmembranen - chara - charophyta - fotosynthese - plantenweefsels - celfysiologie - cell membranes - chara - charophyta - photosynthesis - plant tissues - plasma membranes - cell physiology
    Chlorophyll a fluoresence induction and changes in the electrical potential of the cellular membranes of green plant cells
    Vredenberg, W.J. - \ 1970
    Wageningen : [s.n.] (Publikatie / Centre for Plant physiological research no. 99) - 10
    chlorofyl - cytologie - histologie - plantenfysiologie - planten - celfysiologie - chlorophyll - cytology - histology - plant physiology - plants - cell physiology
    The permeability of dead plant cells for some enzymes
    Gorin, N. - \ 1969
    Wageningen University. Promotor(en): C. den Hartog. - Wageningen : Veenman - 93
    membranen - transport - cytologie - plantenfysiologie - enzymen - enzymologie - fermentatie - celfysiologie - membranes - transport - cytology - plant physiology - enzymes - enzymology - fermentation - cell physiology

    The penetration of α-chymotrypsin and/or pancreatic lipase into dead cells of soybean cotyledons, of yeast and of algae was studied using the enzymic activity as a parameter. In addition a fluorescent antibody technique was applied for the localization of α-chymotrypsin within the soybean cells.

    The digestibility of unheated, EDTA-treated substrate was similar to that of the heated substrate. Since EDTA-treatment increases the permeability of the cell wall and cell membrane of the plant cell without denaturing proteins contained in the cells, we concluded that heating affected mainly the barrier formed by the cell wall, thus permitting a better passage of big molecules (enzymes).

    Leakage of β-amylase (mol. weight 61,700) from soybean occurred only after treatment with EDTA. This confirmed that EDTA enhances the permeability of the cell wall.

    α-Chymotrypsin (mol. weight 24,000) and pancreatic lipase (mol. weight 38,000) penetrated unheated sections, whereas amylopectin (mol. weight 50,000-1,000,000) did not penetrate unheated cotyledons. Apparently the greater dimension of amylopectin compared with α-chymotrypsin and lipase accounts for its lack of entry into unheated soybean cotyledons. However, when we applied the fluorescent antibody technique to localize α-chymotrypsin within the cells from unheated sections, we used antibodies (γ-globulins) having a mol. weight of 150,000. They entered the unheated sections and produced a positive result of the experiment. Consequently the size of the molecules as indicated by the molecular weight, is not the reason why amylopectin did not penetrate the unheated cotyledons. Moreover, this shows that α-chymotrypsin probably opens a way for the entry of globulins. Therefore the entry of proteolytic enzymes is not an inert process. This is conceivable as the enzymes are big molecules with catalytic activity.

    Unheated non-EDTA-treated soybean (with plasmodesmata) was slightly penetrated by α-chymotrypsin, whereas unheated non-EDTA-treated yeast and algae (both without plasmodesmata) were not penetrated at all. Furthermore, in soybean material (unheated or heated) α-chymotrypsin enhanced the penetration of lipase; this effect was absent in the case of yeast. The results obtained strongly suggest that plasmodesmata are a way in dead plant cells for penetration of enzymes.

    Surface - active chemicals as regulators of plant growth, membrane permeability, and resistance to freezing
    Kuiper, P.J.C. - \ 1967
    Wageningen : [Veenman] (Mededelingen Landbouwhogeschool 67-3) - 23
    peulvruchten - zaadkieming - kiemrust - oppervlaktespanningsverlagende stoffen - oogstschade - beschadigingen door vorst - plantenfysiologie - plantengroeiregulatoren - indicatorplanten - celfysiologie - grain legumes - seed germination - seed dormancy - surfactants - crop damage - frost injury - plant physiology - plant growth regulators - indicator plants - cell physiology
    Handleiding voor squashmethoden bij het cytologisch onderzoek aan celdeling in planten
    Christiansen, H. - \ 1965
    Wageningen : Stichting voor Plantenveredeling - 70
    plantenweefsels - cytologie - histologie - celdeling - voortplanting - celcyclus - chromosomen - plantkunde - celfysiologie - plant tissues - cytology - histology - cell division - reproduction - cell cycle - chromosomes - botany - cell physiology
    Verslag van een cytogenetische studie op het laboratorium van dr. Hagberg, Swedish seed association te Svaloef in Zweden, alsmede van bezoeken ... : Januari - februari 1960
    Zeilinga, A.E. - \ 1960
    Wageningen : [s.n.] (Rapport / Instituut voor de Veredeling van tuinbouwgewassen no. 5-6)
    cytologie - plantenfysiologie - voortplanting - celfysiologie - cytology - plant physiology - reproduction - cell physiology
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