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  • 1
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Company
    Nature biotechnology 10 (1992), S. 1436-1445 
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] There are marked differences in the pattern of host gene expression in incompatible plant : microbial pathogen interactions compared with compatible interactions, associated with the elaboration of inducible defenses. Constitutive expression of genes encoding a chitinase or a ribosome-inactivating ...
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  • 2
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] Chalcone synthase (CHS) catalyzes the key regulatory step in the synthesis of a class of plant natural products that function as antibiotics (phytoalexins). Inoculation of leaves of transgenic tobacco containing a CHS promoter-β-glucuronidase (GUS) reporter gene fusion with the bacterium ...
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  • 3
    ISSN: 1432-2048
    Keywords: Glycoprotein (plasma membrane) ; Nicotiana (plasma membrane) ; Plasma membrane (properties)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Monoclonal antibody PN 16.4B4 reacts with an epitope present on the external face of the plasma membrane as shown by immunofluorescent staining of Nicotiana glutinosa L. protoplasts (Norman et al. 1986, Planta 167, 452–459). We show here that this epitope is present in a glycan moiety and defines a family of surface glycoproteins with molecular masses in the range 135–180 kilodalton (kDa). These glycoproteins are exclusively associated with the plasma membrane as demonstrated by immunostaining of highly purified plasma membrane vesicles obtained by aqueous two-phase partitioning of microsomal fractions. The bulk of these glycoproteins were not released by high-salt washing, sonication or hypotonie shock treatment of plasma membrane vesicles, demonstrating a tight association with the membrane. Triton X-114 partitioning of plasma membrane vesicles indicates that these antigens are hydrophilic, peripheral membrane glycoproteins. The glycoproteins were purified by immunoaffinity chromatography following solubilization in sodium dodecyl sulfate and shown to contain glycan moieties abundant in arabinose and galactose linked to a 50-kDa polypeptide rich in alanine, glycine, serine and threonine.
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  • 4
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sites hypersensitive to digestion by DNase I have been identified within the 5′-flanking and 3′-coding sequences of genes encoding the defense enzyme chalcone synthase in bean (Phaseolus vulgaris L.). Two of the 5′-flanking hypersensitive sites are markedly induced upon elicitation of cells with glutathione and delineate sequence elements that are also present in the promoters of coordinately regulated genes. In contrast, other hypersensitive sites within the 5′-flanking sequences are expressed constitutively and one maps within an element that is also present in the promoters of coordinately regulated genes. These results suggest that the transcriptional activation of chalcone synthase genes is accompanied by structural changes in the chromatin associated with the proximal region of the promoter and that these probably reflect the binding of transcription factors tocis-regulatory elements.
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  • 5
    ISSN: 1432-203X
    Keywords: Medicago sativa L. ; Protoplast ; Chalcone synthase ; Promoter ; Transient assay ; Antisense RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have investigated conditions for the uptake and expression of chimeric genes in protoplasts of alfalfa (Medicago sativa L.). Constructs containing the bacterial reporter gene chloramphenicol acetyltransferase (CAT) under the control of either the cauliflower mosaic virus 35S promoter or a bean chalcone synthase (CHS) promoter were introduced into protoplasts by electroporation in the presence of polyethyleneglycol. The extent of expression in the absence of added inducers depended on the conditions for isolation, electroporation and subsequent culture of the protoplasts. Expression of the CHS promoter construct was increased on exposure of the protoplasts to a fungal elicitor or reduced glutathione. The relative levels of induced expression in relation to either basal expression or the type of elicitor used depended on the age of the suspension cultures from which the protoplasts were isolated. Electroporation of protoplasts with a construct from which bean CHS antisense transcripts were synthesized under the control of the 35S promoter resulted in the inhibition of appearance of elicitor-induced endogenous alfalfa CHS activity. The suitability of the alfalfa protoplast system for analysis and potential identification of defense response genes is discussed.
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  • 6
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell suspension cultures of bean (Phaseolus vulgaris) cv. Imuna accumulated isoflavonoid phytoalexins on exposure to elicitor from the phytopathogenic fungus Colletotrichum lindemuthianum (CL). This was preceeded by rapid increases in the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). However, the patterns of expression of PAL and CHS genes differed from those observed in cultures of a previously studied bean cultivar. The relative levels of transcripts from individual members of the CHS multigene family differed significantly at 1.5 h compared to 22.5 h after elicitation. More strikingly, three PAL genes were expressed in cultivar Imuna in response to fungal elicitor, whereas two are expressed in elicitor-treated cell cultures of cultivar Canadian Wonder.
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  • 7
    ISSN: 1432-2048
    Keywords: Cell suspension culture ; Chalcone isomerase ; Elicitor ; Enzyme induction ; Phaseolus (chalcone isomerase) ; Phytoalexin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI〉32 h in control cultures was determined by labelling with 2H from 2H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.
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  • 8
    ISSN: 1573-5028
    Keywords: chalcone synthase ; cis elements ; Phaseolus vulgaris ; seed development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5′ to 3′) in the 5′ region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-β-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position-173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the-326 to-173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position-173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.
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  • 9
    ISSN: 1573-5028
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 10
    ISSN: 1573-5028
    Keywords: elicitor ; gene family ; gene sequence ; Phaseolus vulgaris ; phenylalanine ammonia-lyase ; phenylpropanoid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) genomic sequences were isolated from bean (Phaseolus vulgaris L.) genomic libraries using elicitor-induced bean PAL cDNA sequences as a probe. Southern blot hybridization of genomic DNA fragments revealed three divergent classes of PAL genes in the bean genome. Polymorphic forms were observed within each class. The nucleotide sequences of two PAL genes, gPAL2 (class II) and gPAL3 (class III), were determined. gPAL2 contains an open reading frame encoding a polypeptide of 712 amino acids, interrupted by a 1720 bp intron in the codon for amino acid 130. gPAL3 encodes a polypeptide of 710 amino acids showing 72% similarity with that encoded by gPAL2, and contains a 447 bp intron at the same location. At the nucleotide level, gPAL2 and gPAL3 show 59% sequence similarity in exon I, 74% similarity in exon II, and extensive sequence divergence in the intron, 5′ and 3′ flanking regions. S1 nuclease protection identified transcription start sites of gPAL2 and gPAL3 respectively 99 bp and 35 bp upstream from the initiation codon ATG, and showed that gPAL2 but not gPAL3 was activated by elicitor, whereas both were activated by wounding of hypocotyls. The 5′ flanking region of both genes contain TATA and CAAT boxes, and sequences resembling the SV40 enhancer core. gPAL2 contains a 40 bp palindromic sequence and a 22 bp motif that are also found at similar positions relative to the TATA box in 5′ flanking regions of other elicitor-induced bean genes.
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