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Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake
Middleton, Alistair M. ; Bosco, Cristina Dal; Chlap, Phillip ; Bensch, Robert ; Harz, Hartmann ; Ren, Fugang ; Bergmann, Stefan ; Wend, Sabrina ; Weber, Wilfried ; Hayashi, Ken Ichiro ; Zurbriggen, Matias D. ; Uhl, Rainer ; Ronneberger, Olaf ; Palme, Klaus ; Fleck, Christian ; Dovzhenko, Alexander - \ 2018
Cell Reports 22 (2018)11. - ISSN 2211-1247 - p. 3044 - 3057.
auxin - auxin flux - auxin sensor - endoplasmic reticulum - fluorescent aux - mathematical modeling - microscopy - nucleus - protoplasts - single cells
In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells. Middleton et al. study how the plant phytohormone auxin enters the nucleus by using quantitative phenotyping in single plant cell systems and bespoke mathematical models that relate controlled perturbations to experimentally measurable responses. Their findings show that auxin predominantly enters the nucleus via the endoplasmic reticulum.
The effect of endogenous hydrogen peroxide induced by cold treatment in the improvement of tissue regeneration efficiency
Szechynska-Hebda, M. ; Skrzypek, E. ; Dabrowska, G. ; Wedzony, M. ; Lammeren, A.A.M. van - \ 2012
Acta Physiologiae Plantarum 34 (2012)2. - ISSN 0137-5881 - p. 547 - 560.
oxidative stress - somatic embryogenesis - ascorbate peroxidase - superoxide-dismutase - gene-expression - anther culture - plant-regeneration - winter-wheat - bread wheat - protoplasts
We propose that oxidative stress resulting from an imbalance between generation and scavenging hydrogen peroxide contributes to tissue regeneration efficiency during somatic embryogenesis of hexaploid winter wheat (Triticum aestivum cv. Kamila) and organogenesis of faba bean (Vicia faba ssp. minor cv. Nadwislanski). Endogenous hydrogen peroxide content and antioxidant capacity of cells were determined in initial explants and callus cultures derived from these explants. Regeneration-competent explants (immature embryos) contained more endogenous H2O2 than explants initiated from regeneration-recalcitrant tissue (mature wheat embryos and faba bean epicotyls). Higher H2O2 levels were observed despite the higher activity of antioxidative enzymes (superoxide dismutase and catalase) and the induction of their gene expression. Calli originating from immature embryos retained the capacity of the initial explants: high H2O2 production was observed during the whole culture period. Low temperature treatment (4°C) was found to be an effective factor, which improved both regeneration ability and H2O2 production. Exogenous application to the medium of H2O2 and catalase blocker (3-aminotriazole), but not FeEDTA and superoxide dismutase blocker (diethyldithiocarbamate), also resulted in the enhancement of regeneration efficiency. These results clearly indicate that plant regeneration is specifically regulated by endogenous H2O2 and by factors, which improve its accumulation. Moreover, a study of the activity of various SOD isoforms suggests that not only the absolute concentration of H2O2, but also its localisation might be responsible for controlling regeneration processes
ENOD40 affects elongation growth in tobacco Bright Yellow-2 cells by alteration of ethylene biosynthesis kinetics
Ruttink, T. ; Boot, K. ; Kijne, J. ; Bisseling, T. ; Franssen, H. - \ 2006
Journal of Experimental Botany 57 (2006)12. - ISSN 0022-0957 - p. 3271 - 3282.
rhizobium-legume interaction - soybean nodule development - white clover - expression - protoplasts - division - gene - arabidopsis - nodulation - patterns
Plant developmental processes are controlled by co-ordinated action of phytohormones and plant genes encoding components of developmental response pathways. ENOD40 was identified as a candidate for such a plant factor with a regulatory role during nodulation. Although its mode of action is poorly understood, several lines of evidence suggest interaction with phytohormone response pathways. This hypothesis was investigated by analysing cytokinin-, auxin-, and ethylene-induced responses on cell growth and cell division in transgenic 35S:NtENOD40 Bright Yellow-2 (BY-2) tobacco cell suspensions. It was found that cell division frequency is controlled by the balance between cytokinin and auxin in wild-type cells and that this regulation is not affected in 35S:NtENOD40 lines. Elongation growth, on the other hand, is reduced upon overexpression of NtENOD40. Analysis of ethylene homeostasis shows that ethylene accumulation is accelerated in 35S:NtENOD40 lines. ENOD40 action can be counteracted by an ethylene perception blocker, indicating that ethylene is a negative regulator of elongation growth in 35S:NtENOD40 cells, and that the NtENOD40-induced response is mediated by alteration of ethylene biosynthesis kinetics.
Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus
Pouwels, J. ; Velden, T. van der; Willemse, J. ; Borst, J.W. ; Lent, J.W.M. van; Bisseling, T. ; Wellink, J.E. - \ 2004
Journal of General Virology 85 (2004)12. - ISSN 0022-1317 - p. 3787 - 3796.
green fluorescent protein - resonance energy-transfer - gfp-fusion proteins - fret microscopy - living cells - m-rna - intracellular-distribution - lateral mobility - plant-cells - protoplasts
Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein.
Studies on the C-terminus of the Cowpea mosaic virus movement protein
Bertens, P. ; Heijne, W. ; Wel, N. van der; Wellink, J.E. ; Kammen, A. van - \ 2003
Archives of Virology 148 (2003). - ISSN 0304-8608 - p. 265 - 279.
green fluorescent protein - clover mottle virus - insect cells - tubular structures - subcellular-localization - nsm protein - in-vivo - b-rna - identification - protoplasts
Cowpea mosaic virus (CPMV) spreads from cell-to-cell as virus particles through tubular structures in modified plasmodesmata which are composed of viral movement protein (MP). Mutational analysis of the MP has revealed that the N-terminal and central regions of the MP are involved in tubule formation and that the C-terminal domain probably has a role in the interactions with virus particles. By constructing C-terminal deletion mutants and comoviral hybrid MPs, it was possible to delineate the C-terminal border of the tubule-forming domain to a small region between amino acids 292 and 298. Experiments with tripartite viruses in protoplasts indicated that the C-terminus of the MP is involved in the incorporation of virus particles in the tubule and that for efficient incorporation of virus particles all MP molecules incorporated in a tubule need to contain a functional C-terminus. A mutant virus coding for a MP in which the last 10 C-terminal amino acids were replaced by the green fluorescent protein (GFP) was able to form tubules in protoplasts. These tubules did not contain virus particles, probably because the GFP interferes with the incorporation of virions into the tubule. These results suggest a model for the structure of the tubule in which the C-terminus of the MP is located inside the tubular structure, where it is able to interact with virus particles.
The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein
Carvalho, C.M. ; Wellink, J.E. ; Ribeiro, S.G. ; Goldbach, R.W. ; Lent, J.W.M. van - \ 2003
Journal of General Virology 84 (2003). - ISSN 0022-1317 - p. 2271 - 2277.
tubular structures - rna - protoplasts - cells
Cowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the IMP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified IMP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the IMP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.
Identification of distinct steps during tubule formation by the movement of protein of Cowpea mosaic virus
Pouwels, J. ; Kornet, N. ; Bers, N.E.M. ; Guighelaar, T. ; Lent, J.W.M. van; Bisseling, T. ; Wellink, J.E. - \ 2003
Journal of General Virology 84 (2003). - ISSN 0022-1317 - p. 3485 - 3494.
green fluorescent protein - m-rna - mutational analysis - insect cells - coat protein - protoplasts - plants - chain - involvement - cerulenin
The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement.
Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions
Gopinath, K. ; Bertens, P. ; Pouwels, J. ; Marks, H. ; Lent, J.W.M. van; Wellink, J.E. ; Kammen, A. van - \ 2003
Archives of Virology 148 (2003). - ISSN 0304-8608 - p. 2099 - 2144.
to-cell trafficking - endoplasmic-reticulum - tubular structures - 3a protein - m-rna - protoplasts - plasmodesmata - infection - plants - localization
Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced the formation of fluorescent tubular structures, which shows that subcellular targeting and tubule formation are not affected by fusion of GFP to the C-terminus of the MP. In plants, MPfGFP infections were mostly confined to single epidermal cells and failed to achieve a systemic infection, probably because the fusion of GFP to the MP interfered with MP-virion interaction. MP:GFP mainly accumulated in fluorescent spots in the cell wall of epidermal cells of inoculated leaves, which may represent short tubular structures in modified plasmodesmata. At the cuticle-side of epidermal cells tubular structures were detected indicating that tubule formation in plants, as in protoplasts, does not require the presence of functional plasmodesmata. Furthermore, results were obtained which indicate that CPMV MP:GFP is able to traffic from cell-to-cell by itself. The possible significance of this finding is discussed.
The cytoskeleton and the secretory pathway are not involved in targeting the cowpea mosaic virus movement protein to the cell periphery
Pouwels, J. ; Krogt, G.N.M. van der; Lent, J. van; Bisseling, T. ; Wellink, J.E. - \ 2002
Virology 297 (2002). - ISSN 0042-6822 - p. 48 - 56.
green fluorescent protein - endoplasmic-reticulum - tubular structures - m-rna - intracellular-distribution - plasma-membrane - plant - plasmodesmata - protoplasts - microtubules
The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway, Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A, severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs, (C) 2002 Elsevier Science (USA).
The role of NSm during tomato spotted wilt virus infection
Storms, M.M.H. - \ 1998
Agricultural University. Promotor(en): R.W. Goldbach; J.W.M. van Lent. - S.l. : Storms - ISBN 9789054859215 - 118 p.
bunyaviridae - plantenvirussen - plantenziekteverwekkers - infectie - beweging - eiwitten - protoplasten - plant viruses - plant pathogens - infection - movement - proteins - protoplasts
<p>In the past ten years the genome organisation of tomato spotted wilt virus (TSWV) has been intensively studied in our laboratory. Complete genome sequence data revealed that this enveloped plant virus belongs to the Bunyaviridae, a virus family further restricted to animals.</p><p>Hence, TSWV is a splendid model to investigate which viral encoded factors are needed for a virus to successfully infect a plant. Comparison of the genome of TSWV with those of the animal-infecting members of the Bunyaviridae reveals the presence of an extra cistron in the TSWV genome. This cistron encodes a non-structural protein of 33.6 kDa, denoted NS <sub>M</sub> . Although some animal infecting Bunyaviridae also specify a protein referred to as NS <sub>M</sub> , this polypeptide does not represent a separate gene product. Hence, the extra NS <sub>M</sub> gene may very well represent the key function for genetic adaptation of Bunyaviruses to plant hosts. For a successful infection of plants, viruses have to pass the plant specific cell wall barrier through plasmodesmata. As the size of most viruses, and even their genomes, exceeds the physical space provided by plasmodesmata for intercellular transport of macromolecules (the size exclusion limit or SEL), cell-cell movement requires a structural modification of the plasmodesmata. For a number of plant viruses it has been shown that they encode one or more movement proteins (MPs) which achieve viral cell-cell movement, among others by modification of the plasmodesmata, most likely in co-operation with host factors and/or other viral proteins.</p><p>At the onset of this PhD research, the working hypothesis was that NS <sub>M</sub> could very well represent the MP of TSWV. This was also based on the presence of a conserved sequence in the NS <sub>M</sub> protein, the so-called "D-motif", characteristic for several other plant viral MPs.</p><p>To obtain experimental data for the role of NS <sub>M</sub> in the TSWV infection cycle, first the expression kinetics and intracellular behaviour of the protein were studied in systemically infected <em>Nicotiana rustica</em> leaf tissue. The NS <sub>M</sub> gene was cloned and expressed in <em>E. coli</em> , the protein was purified and a specific antiserum made. Time course analyses of systemically infected <em>N. rustica</em> leaf tissue, using this anti-NS <sub>M</sub> serum, revealed an early and transient presence of the protein in infected cells, a manifestation that is unique for NS <sub>M</sub> and not found for other viral proteins of TSWV (Chapter 2). The early and temporary appearance of NS <sub>M</sub> suggested that the protein has an early function in the infection cycle in concert with the idea that it is involved in cell-cell movement. During infection of a cell, NS <sub>M</sub> was first localised to newly formed viral nucleocapsids in the cytoplasm and plasmodesmata. Moreover, NS <sub>M</sub> was found to assemble into tubular structures that penetrate the plasmodesmata in a unidirectional way. These tubules were also formed upon expression of the cloned NS <sub>M</sub> gene in protoplasts (Chapter 3)</p><p>The formation of similar tubular structures has been shown to be an essential step during cell-to-cell movement of a number of RNA (e.g. como- and nepoviruses) and DNA (caulimoviruses) plant viruses. For these latter viruses, it was shown that mature virions are transported from cell-to-cell through MP-induced tubular structures. Investigations on the contents of the TSWV tubules, formed on synchronously infected protoplasts, revealed the presence of only the nucleoprotein inside the tubular structures (Chapter 4). Based on this, it is tempting to assume that TSWV is translocated into the adjacent non-infected cell as non-enveloped, viral nucleocapsids.</p><p>An important characteristic of several plant virus MPs is that they evoke a modification of the macromolecular diffusion limits (SEL) of plasmodesmata. To investigate whether NS <sub>M</sub> has a similar function, the NS <sub>M</sub> gene was transgenically expressed in <em>Nicotiana tabacum</em> SR1 plants (Chapter 5). The resulting transgenic plants were examined for NS <sub>M</sub> expression, subcellular localisation of this protein, and changes in the diffusion properties of the plasmodesmata. The NS <sub>M</sub> expression levels obtained were invariably low. However, the NS <sub>M</sub> protein could be localised to over 80% of the plasmodesmata in various transgenic tissues, demonstrating its specific targeting to and association with these organelles. Probably as a result of NS <sub>M</sub> accumulation at the plasmodesmata, the plants showed severe aberrations in growth and development (Chapter 5).</p><p>Possible alterations in plasmodesmal functioning were further analysed by microinjection of fluorescent probes of various sizes into mesophyll cells of the NS <sub>M</sub> transgenic plants. Two different methods of microinjection were used and compared, one by which the influx of probes was achieved by a pressure pulse and a second, by diffusion (iontophoresis). Pressure injection studies on NS <sub>M</sub> transgenic plants with fluorescent probes of different molecular mass showed an increase in plasmodesmal SEL similar to that of control transgenic plants expressing the TMV MP. As, in this respect, NS <sub>M</sub> modifies the plasmodesmal function in a similar manner as the TMV MP, NS <sub>M</sub> was concluded to be the tospoviral MP. Strikingly, when iontophoresis was performed on NS <sub>M</sub> - or TMV-MP transgenic plants, the plasmodesmata showed a decrease rather than an increase in SEL. In control (non-transgenic) plants, the outcome of pressure injection and iontophoresis was identical. This strongly suggested that accumulation of viral MP made the cells respond differently to the microinjection methods used.</p><p>A possible interpretation for these remarkable differences is that TSWV and TMV MP, inhibits or partly blocks, the communication pathway for macromolecular trafficking, as recorded by the less invasive iontophoresis method, which also explains the disturbed morphology and physiology of the MP transgenic plants. The protein barrier at the plasmodesmata may be overcome by a pressure pulse, thereby revealing an increase of SEL induced by the plant viral MP (Chapter 6).</p><p>As TSWV also replicates in its thrips vectors, it was of interest to explore whether NS <sub>M</sub> also had a function during the infection cycle in the insect. The expression kinetics and localisation of the NS <sub>M</sub> protein was studied in all developmental stages of the major viral vector, the thrips <em>Frankliniella occidentalis</em> (Chapter 8). Besides the <em>in situ</em> analyses of NS <sub>M</sub> in the vector itself, the behaviour of the protein was also investigated in heterologous <em>Spodoptera frugiperda</em> and <em>Trichoplusia ni</em> insect cell cultures and in baby hamster kidney (BHK21) cells, a mammalian cell system unrelated to the natural hosts of TSWV (plants and insects)(Chapter 7). Upon expression of NS <sub>M</sub> in cultured insect cells, the protein specifically targeted to the cell periphery and formed tubular structures at the cell surface in a similar fashion as found in plant protoplasts. However, in mammalian cells no plasmamembrane targeting or tubular structures were found. The results obtained for insect cells indicate that the NS <sub>M</sub> protein has the potential to form tubular structures in the absence of any plant specific component.</p><p>In the vector <em>F. occidentalis</em> , NS <sub>M</sub> was present at only low levels in mostly midgut epithelium cells of L2 larvae and in salivary gland cells and midgut muscle cells of adult thrips. Characteristic structures or associations as found in infected plants (e.g. tubule formation, association with viral nucleocapsids) could not be discerned in any of the developmental stages of the thrips. Although this does not exclude a function for NS <sub>M</sub> in the TSWV vector, this function would then be unrelated to its activity as MP.</p><p>In summary, the investigations described in this thesis all demonstrate that NS <sub>M</sub> represents the MP of TSWV, and that it mediates the cell-cell movement of presumably non-enveloped viral nucleocapsids through transiently produced tubular structures that penetrate plasmodesmata. As such, the extra NS <sub>M</sub> gene in the genome of TSWV indeed encodes the key protein required for pathogenicity of Bunyaviridae towards plants.</p>
|Protoplastregeneratie en somatische hybridisatie bij lelie en tulp : eindverslag van het project Protoplastregeneratie en somatische hybridisatie bij lelie en tulp
Famelaer, I.D.H. ; Ennik, E. ; Creemers - Molenaar, J. - \ 1996
Wageningen : CPRO-DLO (Urgentieprogramma bollenziekte- en veredelingsonderzoek ) - 30 p.
hybriden - soortkruising - liliaceae - sierplanten - onderzoek - somatische hybridisatie - verwijderde hybridisatie - lilium - tulipa - protoplasten - hybrids - interspecific hybridization - ornamental plants - research - somatic hybridization - wide hybridization - protoplasts
|Transformation and parasexual recombination in Fusarium oxysporrum f.sp. gladioli
Bao, J.R. - \ 1995
Lisse : Laboratorium voor Landbouwkundig Onderzoek (Intern LBO-rapport nr. 043)
gladiolus - plantenziekteverwekkers - recombinatie - genetische transformatie - protoplasten - fusarium oxysporum f.sp. gladioli - plant pathogens - recombination - genetic transformation - protoplasts
Studies on genetic transformation of coffee by using electroporation and the biolistic method
Boxtel, J.H.J. van - \ 1994
Agricultural University. Promotor(en): Evert Jacobsen; A.B. Eskes. - S.l. : Van Boxtel - ISBN 9789054853152 - 125 p.
coffea - koffie - protoplasten - recombinant dna - electroporatie - biolistiek - coffee - protoplasts - electroporation - biolistics
<p>The present study aimed simultaneously at an improvement of coffee regeneration systems and at a definition of factors influencing the efficiency of direct gene transfer methods. The development of an improved regeneration system, based on high frequency somatic embryogenesis from leaf explants, passing through multiplication of embryogenic callus in liquid medium, is described. This method can contribute to the obtaining of high protoplast yields and offers perspectives for use in genetic transformation systems of coffee. Several factors affecting protoplast isolation, electroporation and regeneration were studied. This system appeared to be appropriate for transient expression studies but, due to difficulties with protoplast regeneration, less promising for achieving stable expression. Further expression studies were performed using particle gun bombardment on different tissues of several coffee genotypes. Best results were obtained using <em>in vitro</em> cultured leaves of <em>Coffea arabica</em> and plasmids carrying the EF1α-A1 promoter of <em>Arabidopsis thaliana.</em> The effect of tungsten particles on callus induction and the fate of GUS-expressing cells after bombardement on leaves has been described, and the consequences for their use are discussed. Studies on five selective agents showed best prospects of the herbicide glufosinate for detection of stably transformed coffee tissue. It was concluded that avoidance of polyphenolic oxidation, caused by tissue wounding, is of great importance for the development of a reliable genetic transformation method for coffee.
Plant protoplasts as a model system to study phytochrome-regulated changes in the plasma membrane
Bossen, M.E. - \ 1990
Agricultural University. Promotor(en): W.J. Vredenberg; R.E. Kendrick. - S.l. : Bossen - 100 p.
plantenfysiologie - fotosynthese - protoplasten - cellen - celmembranen - plant physiology - photosynthesis - protoplasts - cells - cell membranes
<p><TT>Protoplasts, isolated from the primary leaves of dark-grown wheat ( <em>Triticum aestivum</em> L.), have been used as a model system to study phytochrome-regulated changes of the plasma membrane. Such protoplasts only swelled after red light (R)-irradiation, when Ca</TT><sup>2+</SUP><TT>was present in the medium. Far-red light (FR), after R, prevented swelling, indicating phytochrome involvement. Swelling was inhibited when La</TT><sup>3+</SUP><TT>or the Ca <sup>2+</SUP>-channelblocker Verapamil were added. Swelling was induced in darkness by the Ca <sup>2+</SUP>-ionophore A23187 and the calmodulin antagonist W <sub>7</sub> . It is proposed that Rirradiation leads to opening of Ca <sup>2+</SUP>-channels, resulting in an increase of the cytoplasmic [Ca <sup>2+</SUP>] and protoplast swelling.</TT><p><TT>The effect of modulators of G-proteins and the phosphatidylinositol cycle, as known in animal cells, on the swelling response was examined. The R-induced swelling was inhibited by GDP-β-S and by neomycin, Li <sup>+</SUP>and H <sub>7</sub> . In darkness, swelling was found when GTP-γ-S or PMA were added to the protoplasts. All agonists and antagonists used, influenced the swelling response, as predicted by transposition of the animal model to plants. This suggests that R-irradiation, leads to activation of a G- protein, which results in the opening of Ca <sup>2+</SUP>-channels.</TT><p><TT>Plant hormones also induced protoplast swelling in the presence of Ca <sup>2+</SUP>, while swelling was inhibited by GDP-β-S Acetylcholine induced, contrary to R-irradiation, swelling in the absence of Ca <sup>2+</SUP>, when K <sup>+</SUP>or Na <sup>+</SUP>were present in the medium. This swelling was not inhibited by GDP-β-S</TT><br/> <p><TT>The Ca <sup>2+</SUP>-sensitive dye murexide, has been used to monitor phytochrome-regulated changes in the [Ca <sup>2+</SUP>] of the medium. Red light induced a Ca <sup>2+</SUP>-efflux, while FR reversed this effect. The R-induced efflux was inhibited by Verapamil and W <sub>7</sub> by approx. 75%. Therefore, the efflux, via a Ca <sup>2+</SUP>-ATPase, appears to be dependent on the activation of Ca <sup>2+</SUP>-channels and a Ca <sup>2+</SUP>-influx.</TT><p><TT>The fluidity of the protoplast plasma membrane was studied, using the fluorescent membrane probe DPH. After R the anisotropy of DPH (r <sub>f</sub> ) was higher, indicating a decrease in membrane fluidity. In darkness, r <sub>f</sub> also increased upon osmotically induced protoplast swelling. It is not clear, whether R causes changes in membrane fluidity, independent of changes in volume.</TT><p><TT>The observed changes in plasma membrane properties after R-irradiation, show that protoplasts are an useful tool for studying phytochrome action in higher plants.</TT>
|Verslag van studiereis naar Polen, 21 - 18 oktober 1984
Dons, J.J.M. - \ 1985
Wageningen : IVT (Rapport / Instituut voor de Veredeling van Tuinbouwgewassen no. 203) - 5 p.
celkweek - cellen - komkommers - cucumis sativus - embryokweek - meristemen - polen - protoplasten - onderzoek - weefselkweek - cell culture - cells - cucumbers - embryo culture - meristems - poland - protoplasts - research - tissue culture
|Cultuur van protoplasten van sla, lactuca : IVT Wageningen, 1 december 1983 - 31 december 1984
Hogenboom, N.M. - \ 1985
Wageningen : IVT (Rapport / Instituut voor de Veredeling van Tuinbouwgewassen 208) - 11 p.
cellen - embryokweek - genetica - lactuca sativa - slasoorten - protoplasten - weefselkweek - cells - embryo culture - genetics - lettuces - protoplasts - tissue culture
In dit verslag worden methodes beschreven voor isolatie van protoplasten van sla. Omdat regeneratie van deze protoplasten nog niet mogelijk bleek kon het meer praktisch gerichte onderzoek, zoals de overdracht van genen d.m.v. somatische hybridisatie en de beoordeling van de bruikbaarheid van somaklonale variatie, nog niet worden gestart. Wel worden enkele inleidende onderzoekjes beschreven over de isolatie van protoplasten uit de wilde slasoorten en een methode voor het verkrijgen van witte slaplanten die als fusiepartner gebruikt kunnen worden
|Isolatie en regeneratie van protoplasten van komkommer (Cucumis sativus)
Dons, J.J.M. - \ 1984
Wageningen : IVT (Rapport / Instituut voor de Veredeling van Tuinbouwgewassen no. 202) - 9 p.
celkweek - cellen - komkommers - cucumis sativus - meristemen - protoplasten - onderzoek - weefselkweek - cell culture - cells - cucumbers - meristems - protoplasts - research - tissue culture