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  • 1
    ISSN: 1573-5028
    Keywords: elicitation ; gene transcription ; isoflavonoid phytoalexins ; Medicago sativa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nuclear transcript run-on analysis was used to investigate the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures. Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10–20 min after elicitation. Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-O-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate. Transcription of chalcone 2′-O-methyltransferase (CHOMT), and 1,3-β-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively. Treatment of cells with the PAL inhibitor L-α-aminooxy-β-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 18 (1992), S. 1189-1190 
    ISSN: 1573-5028
    Keywords: Medicago sativa ; cell culture ; rRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5028
    Keywords: fungal elicitor ; isoflavone reductase mRNA ; Medicago sativa ; phytoalexin biosynthesis ; stereochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The major phytoalexin in alfalfa is the isoflavonoid (−)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (−)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2′-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35400) derived from the 954 bp open reading frame compares favorably to estimated M rs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 19 (1992), S. 319-321 
    ISSN: 1573-5028
    Keywords: Medicago sativa ; cell culture ; protein disulfide isomerase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 213-220 
    ISSN: 1573-5044
    Keywords: Glomus versiforme ; isoflavone reductase ; medicarpin ; Medicago sativa ; phytoalexin ; Phoma medicaginis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed. An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene β-glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in β-glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-9368
    Keywords: Medicago sativa ; chitinase ; glucanase ; transgenic ; plants ; antifungal proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA sequences coding for an acidic glucanase (Aglul) that is expressed in elicited alfalfa cell suspension cultures, and a rice basic chitinase (RCH10) that is induced by elicitor and wounding, were placed into constitutive expression cassettes under control of the cauliflower mosaic virus 35S promoter or 35S enhancer sequences, and introduced in alfalfa plants of the regenerable cultivar Regen SY byAgrobacterium-mediated transformation. Southern and northern blot analysis confirmed stable incorporation and transcription, respectively, of the chimaeric genes in the transgenic plants. Active rice chitinase was expressed in alfalfa leaves, and leaves of plants transformed with theAglul sequence exhibited increased glucanase activity and the appearance of an additional glucanase band on activity gels. A glucanase of similar native electrophoretic mobility was constitutively present in root extracts of non-transformed alfalfa plants, and was induced in pathogen-infected leaves, presumably reflecting the expression pattern of the endogenousAglul gene. Thus, expression of the chimaericAglul transgene increased the amount, and broadened the tissue-type constitutive expression, of theAglul protein compared to control plants. Transgenic alfalfa plants containing a binary vector with both chimaeric genes in tandem expressed each gene to a much lesser extent than transgenic plants containing a single chimaeric gene. Expression of RCH10 in transgenic alfalfa did not appear to affect negatively theRhizobium/alfalfa interaction. Analysis of primary transformants for response to several fungal pathogens of alfalfa indicated statistically significant symptom reduction only in the case ofPhytophthora megasperma f. sp.medicaginis (Pmm), and only in plants overexpressingAglul. Resistance against Pmm segregated with glucanase expression in a cross between transgenic Regen SY and the commercial alfalfa cultivar Apollo.
    Type of Medium: Electronic Resource
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