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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK; Malden, USA : Munksgaard International Publishers
    Physiologia plantarum 122 (2004), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot (Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose-rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3-, 1,6- and 1,3,6- form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Physiologia plantarum 114 (2002), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Arabinogalactan-protein (AGP) epitopes are known to display developmentally regulated patterns of expression in several plant tissues. Therefore, AGPs have been suggested to play a role in plant development. Somatic embryogenesis is regulated by AGPs as well as by EP3 endochitinases. Using four different methods we have analysed the composition of AGPs in immature carrot seeds. The results obtained show that: (1) the native electrophoretic mobility of such AGPs changes during development; (2) AGP epitopes in immature seeds are developmentally regulated; (3) enzymatically released fragments of AGPs show that the composition of these molecules changes as a function of development; and (4) the biological activity of AGPs on the formation of somatic embryos changes depending on the age of the seeds. Our results suggest that degradation of maternally derived AGPs occurs after fertilization, while cellularization of the endosperm leads to synthesis of a new set of AGPs. The presence of an endochitinase cleavage site as well as the capacity to increase somatic embryogenesis only occurred in AGPs that were isolated from seeds in which the endosperm had been cellularized. Apparently, both EP3 endochitinases and somatic embryogenesis-promoting AGPs are developmentally regulated in immature carrot seeds.
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  • 3
    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.
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  • 4
    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.
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  • 5
    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.
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  • 6
    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.
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  • 7
    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.
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  • 8
    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.
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  • 9
    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.
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  • 10
    ISSN: 1432-072X
    Keywords: Thiobacillus A2 ; Mixotrophy ; Competition ; Mixed cultures ; Facultative chemolithotroph ; Ecological niche
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Competition in a chemostat between the versatile Thiobacillus A2 and the specialized T. neapolitanus for thiosulfate as the sole growth-limiting substrate, led to dominance of the specialized over the versatile organism, at dilution rates ≥0.025 h-1. Increasing concentrations of acetate or glycollate in the thiosulfate medium caused increased relative numbers of T. A2 in steady states at D=0.07 h-1. Eventually, with 10–12 mmol of organic substrate per litre, complete dominance of T. A2 over T. neapolitanus occurred. Mixed cultures of T. A2 and a specialized spirillumshaped heterotroph, competing for acetate as sole growth-limiting substrate resulted in complete dominance of the heterotroph at dilution rates of 0.07 and 0.15 h-1. In this case increasing concentrations of thiosulfate in the acetate medium, up to 10 mM, eventually led to the elimination of the heterotroph. These results have been interpreted as evidence that T. A2 was growing mixotrophically. As the concentration of the second substrate was raised, the number of T. A2 cells increased and as a result T. A2 consumed an increasing portion of the common substrate. In mixed chemostat cultures containing all three organisms, T. A2 could maintain itself with all tested ratios of acetate and thiosulfate in the inflowing medium. The heterotroph was excluded from the culture below a relatively low acetate to thiosulfate ratio, whilst above a relatively high acetate to thiosulfate ratio T. neapolitanus was completely eliminated. These results were discussed in relation to the ecological niche of Thiobacillus A2-type organisms.
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