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  • 1
    Electronic Resource
    Electronic Resource
    Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.) (1993)
    Springer
    Molecular and cellular biochemistry 123 (1993), S. 159-166 
    Details
    ISSN: 1573-4919
    Keywords: lipid transfer protein ; plant (carrot) ; fatty acid-binding ; cutin synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2-β-galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01076488
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  • 2
    Electronic Resource
    Electronic Resource
    Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis (1997)
    Springer
    Planta 203 (1997), S. 188-195 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Arabinogalactan protein ; Daucus ; Mono clonal antibody (JIM8 ; ZUM18) ; Somatic embryo genesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Secreted arabinogalactan proteins (AGPs), isolated on the basis of specific epitopes, have been reported that can either enhance (ZUM18 AGP fraction) or inhibit (ZUM15 AGP fraction) carrot (Daucus carota L.) somatic embryo development (Kreuger and van Holst, 1995, Planta 197: 135–141). Here, we report that addition of the ZUM18 AGP fraction to different size-fractionated cell populations isolated from embryogenic carrot suspension cultures does not show a significant effect on the frequency and the morphology of the somatic embryos produced. An AGP fraction containing the JIM8 epitope showed an inhibitory effect on the frequency of somatic embryo development from single cells. Addition of carrot-seed AGPs to non-embryogenic cell suspensions did not directly promote embryogenic competence in the suspension culture. Only after enrichment for cell clusters and removal of most of the single cells was an increase in embryogenic competence observed. These results indicate that cell type composition in suspension cultures is important for evaluating the effect of exogenous AGPs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004250050181
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  • 3
    Electronic Resource
    Electronic Resource
    Description of somatic-embryo-forming single cells in carrot suspension cultures employing video cell tracking (1994)
    Springer
    Planta 194 (1994), S. 565-572 
    Details
    ISSN: 1432-2048
    Keywords: Cell tracking ; Daucus ; Development (single cell) ; 2,4-Dichlorophenoxyacetic acid ; Phytagel (cell immobilisation) ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cell-tracking system was established to determine the capability of individual single suspension cells of carrot (Daucus carota L.) to develop into somatic embryos. When immobilised in phytagel, 127 out of 30 318 single suspension cells smaller than 22 μm in diameter developed into a somatic embryo. Single cells present at the start of the experiment were classified on the basis of their morphology into five groups: small spherical vacuolated cells; small spherical cytoplasm-rich cells; oval vacuolated cells; elongated vacuolated cells and cells that could not be classified into either one of these groups. Single cells of all morphologically distinguishable single cell types developed into somatic embryos with a frequency that varied between 19 and 100 somatic embryos per 10 000 cells. This suggests that the capability of individual single cells to form somatic embryos is not restricted to a particular cell type distinguishable on the basis of its morphology. Three major pathways were observed during development. Oval and elongated cells developed into somatic embryos via an asymmetrical cell cluster. Spherical cells developed via a symmetrical cell cluster into somatic embryos. Before formation of a somatic embryo, cells of a more variable initial morphology first developed aberrantly shaped cell clusters. This suggests that the developmental pathway leading to a somatic embryo can be predicted by the initial single-cell morphology.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00714471
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  • 4
    Electronic Resource
    Electronic Resource
    The secretory nature of the lesion of carrot cell variant ts11, rescuable by endochitinase (1997)
    Springer
    Planta 203 (1997), S. 381-389 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Chitinase ; Daucus ; Plant development ; Secretion ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The carrot cell variant ts11 is unable to form somatic embryos at the non-permissive temperature of 32 °C, but the block can be overcome by the addition of a 32-kDa acidic endochitinase to the medium. In this work we conducted a cyto-histological analysis of the blocked embryo forms. The morphology of the endomembrane system is altered; in particular, the ER is dilated and may show electron-dense precipitates and continuity with the plasma membrane. These morphological alterations do not occur in the presence of externally-added endochitinase. We also noticed modifications of the culture medium that are probably related to the morphological observations: the total amount of secreted proteins is reduced and pulse-chase experiments revealed that, compared with wild-type cells, the secretion of major polypeptides is reduced while new minor polypeptides are secreted. Western blot analysis revealed the presence of the binding protein BiP, a resident of the ER and of glutamine synthase, a cytosolic protein, in the medium of ts11 but not wild-type cells. These results indicate that ts11 is altered in the secretory pathway but do not clarify the role of endochitinase.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004250050204
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  • 5
    Electronic Resource
    Electronic Resource
    Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures (1990)
    Springer
    Plant cell reports 9 (1990), S. 221-223 
    Details
    ISSN: 1432-203X
    Keywords: Calcium ; Daucus carota ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10−3 M were transferred to hormone-free medium containing 10−2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10−4 M calcium were transferred to hormone-free medium with 10−3 M calcium. At calcium concentrations between 6·10−3 and 10−2 M globular stage somatic embryos were found in cultures supplemented with 2·10−6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232184
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  • 6
    Electronic Resource
    Electronic Resource
    Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis (1996)
    Springer
    Planta 200 (1996), S. 167-173 
    Details
    ISSN: 1432-2048
    Keywords: Arabinogalactan protein epitope ; Cell tracking ; Daucus ; Monoclonal antibody (JIM8) ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00208305
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  • 7
    Electronic Resource
    Electronic Resource
    Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings (1982)
    Springer
    Planta 156 (1982), S. 129-135 
    Details
    ISSN: 1432-2048
    Keywords: Etiolation ; Light and mRNA/protein patterns ; mRNA (light-regulated) ; mRNA (organ-specific) ; Pisum ; Protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The diversity of abundant mRNA sequences in various parts of 4-d etiolated pea seedlings (Pisum sativum L. var. Rondo CB) was compared by a cell-free translation of the mRNAs in the presence of [35S]methionine and by an analysis of the products by two-dimensional electrofocussing/ electrophoresis (2D separation). The various parts of the seedlings were also examined for the pattern of protein synthesis in vivo. Proteins were labeled by injection of [35S]methionine into the cotyledons, followed by 2D separation of the products. Over 95% of the abundant mRNA sequences and newly synthesized abundant polypeptides were shared by all parts of etiolated seedlings, including the cotyledons. However, a few distinct differences were observed when comparing mRNAs of roots and shoots; the most prominent among these were a group of six abundant mRNA sequences found exclusively in shoots. Only about 30% of the polypeptides synthesized on isolated RNA could be traced in equivalent positions on the gels as the polypeptides synthesized in vivo. Analysis of total RNA from light-grown pea seedlings showed the appearance of some twenty-five translation products not found with total RNA from etiolated seedlings, while about nine other translation products disappeared. At least ten of the light-induced RNA sequences were also present after growth in low-intensity red light (λ〉600 nm) and are therefore thought to be controlled by the phytochrome system. Comparison of 11-d light-grown pea plants with 4-d light-grown seedlings did not reveal additional translatable RNA sequences, indicating that the major morphogenetic changes that occur after 4 d are not accompanied by significant changes in the pattern of abundant RNA sequences.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00395427
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  • 8
    Electronic Resource
    Electronic Resource
    Sequence diversity of polysomal mRNAs in roots and shoots of etiolated and greened pea seedlings (1983)
    Springer
    Planta 158 (1983), S. 42-50 
    Details
    ISSN: 1432-2048
    Keywords: Genome expression ; mRNA (light-regulated, sequence complexity) ; Ptsum (genome expression)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The sequence complexity and abundance of polysomal mRNA populations of pea seedlings were measured using RNA excess hybridization to both single-copy DNA and complementary DNA. The estimated sequence complexity of the polysomal mRNA populations was 2.5·107 nucleotides or 19,400 different mRNAs of average size. Since the haploid genome size of pea was found to be 4.0·109 nucleotide pairs, only 0.62% of the total haploid genome of pea was transcribed into polysomal mRNA. The roots and shoots of 4-d etiolated and light-grown seedlings contained similar numbers of diverse mRNAs. The RNA excess hybridizations, using single-copy DNA enriched for sequences transcribed in either light-grown shoots or etiolated roots and single-copy DNA depleted of such sequences, indicated that at least 92% of the sequence complexity of polysomal mRNAs was identical in roots and shoots irrespective of the presence of a functional photosynthetic system. In contrast, RNA excess hybridization to complementary DNA revealed that 21% of the polysomal polyadenylated mRNA mass found in light-grown shoots was absent in etiolated roots. The kinetics of these hybridizations indicated that this was due to the appearance of a limited number of abundant mRNAs under conditions of illumination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00395401
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  • 9
    Electronic Resource
    Electronic Resource
    Acquisition of embryogenic potential in carrot cell-suspension cultures (1988)
    Springer
    Planta 176 (1988), S. 196-204 
    Details
    ISSN: 1432-2048
    Keywords: Cell suspension culture ; Daucus ; Embryogenic potential ; Excreted cell factor ; Gene expression mRNA (in vitro translation)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [35S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [35S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00392445
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  • 10
    Electronic Resource
    Electronic Resource
    Gene-expression programs in embryogenic and non-embryogenic carrot cultures (1988)
    Springer
    Planta 176 (1988), S. 205-211 
    Details
    ISSN: 1432-2048
    Keywords: Daucus ; Embryogenic potential ; Gene expression ; mRNA ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Somatic embryogenesis can be synchronized by enriching carrot (Daucus carota L.) suspension cultures for small, dense clusters of cells termed proembryogenic masses (PEMs). Gene-expression programs of PEMs were compared with those of embryonic and mature tissues by in-vitro translation of representative mRNA populations and by nucleic-acid hybridization. Analysis of invitro-translated polypeptides by two-dimensional polyacrylamide gel electrophoresis revealed striking similarities between the mRNA populations of PEM and torpedo-stage embryos; substantial differences, however, were observed when in-vitro translation products of PEMs and torpedo embryos were compared with those of hypocotyls and leaves. Northern blots of RNA isolated from PEMs, staged embryos, and mature carrot tissues were hybridized with cDNA probes for Dc3, Dc5 and Dc13; these cDNA recombinants represent mRNAs that are regulated during carrot somatic embryogenesis. The pattern of expression of these embryo-regulated transcripts was similar in PEMs and somatic embryos but differed in other carrot tissues. These results indicate that many of the molecular processes of embryogenesis are already established in PEMs in the presence of auxin. Additional experiments indicate the utility of Dc3 as a molecular marker for the acquisition of embryogenic potential.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00392446
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