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  • 1
    ISSN: 1432-2048
    Keywords: Arabidopsis (fusicoccin-insensitive) ; Fusicoccin (receptor) ; H+ ; ATPase ; H+-extrusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H+-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a 3H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: glyphosate ; 5-enolpyruvylshikimate 3-phosphate synthase ; in vitro transcription ; overproduction ; enzyme stabilization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell cultures of Corydalis sempervirens, tolerant to the herbicide glyphosate, have a 30–40-fold increased level of the herbicide's target enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a ten-fold enhanced level of the corresponding mRNA but no amplification of the gene (Holländer-Czytko et al., Plant Mol Biol 11 (1988) 215–220). The increase at the transcriptional level is due to a higher rate of transcription of the gene, which was observed in run-off transcription assays with isolated nuclei. The further amplification at the protein level is the result of stabilization of the enzyme by the herbicide. In the presence of glyphosate the half-life of EPSP synthase was doubled leading to higher levels of both protein and enzyme activity. Overproduction of the enzyme in adapted cultures is stable at the transcriptional level, as cells from adapted cultures grown in the absence of glyphosate for three years still display an about ten-fold higher enzyme activity and transcript level than non-adapted cultures.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; allene oxide synthase ; CYP74 ; octadecanoids ; jasmonate biosynthesis ; 12-oxo-phytodienoic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thalina.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-5028
    Keywords: glyphosate ; herbicide ; 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase ; enzyme overproduction ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 mM glyphosate [N-(phosphonomethyl)glycine] display a 30- to 40-fold increase in the cellular content of 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase, the target enzyme of the herbicide. Translatable mRNA activity as well as transcript levels for EPSP synthase were increased 8-to 12-fold in the adapted (glyphosate-tolerant) as compared to the non-adapted (glyphosate-sensitive) cultures. Northern blot analysis revealed a single 1.8 kb transcript after hybridization with an oligonucleotide probe deduced from the N-terminal amino acid sequence of the enzyme. No significant differences in the relative abundance of EPSP synthase-specific DNA sequences could be detected, however, in Southern and dot blot analyses of restricted DNA isolated from the two cultures. We conclude that the overproduction of EPSP synthase in glyphosate-tolerant C. sempervirens cells is not based on the amplification of the corresponding gene.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-2048
    Keywords: Key words: Biological life support ; Ceratophyllum (morphology ; physiology) ; Ecosystem (closed aquatic) ; Nitrate uptake
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The Closed Equilibrated Biological Aquatic System (CEBAS) consists of four subcomponents which form a closed (artificial) aquatic ecosystem initially designed to study the long-term influence of space conditions on several successive generations of aquatic organisms. Teleost fishes and water snails in the zoological component produce CO2, ammonium ions and waste compounds which can be utilized after ammonium is oxidised in a microbial component by the botanical component consisting of a rootless, aquatic higher plant species which eliminates ions, i.e. nitrate, and produces oxygen for animal respiration. An electronic component serves as a data-acquisition and regulation device for temperature and oxygen-dependent illumination of the plant chamber. A comprehensive interdisciplinary research programme, focused around the CEBAS, is especially well developed in the field of zoology. It covers a ground laboratory and preparations for two scheduled spaceflight projects, as well as aspects of combined animal-plant food production modules for human nutrition in bioregenerative space life-support systems and for terrestrial production sites. In the botanical research programme, morphological investigations on Ceratophyllum demersum L. performed with light and electron microscopy have demonstrated a gas lacuna system which, in addition to starch grains in the plastids, might regulate the buoyancy of the plant and/or serve as a `gas skeleton'. Also, a remarkable symmetry in the arrangement of tissues was observed in stems and older leaves. The photosynthetic capacities of Ceratophyllum in the CEBAS-MINI MODULE proved to be more than sufficient for life support, and experiments on nitrate uptake into the plants showed their capacity to utilize ions from the water.
    Type of Medium: Electronic Resource
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