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  • 1
    ISSN: 1573-5028
    Keywords: fatty acid synthesis ; malonyl CoA-ACP transacylase ; seed development ; transgenic rape ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: GTP binding ; ADP ribosylation ; Zea mays ; Escherichia coli ; fatty acid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5028
    Keywords: acyl carrier protein ; Brassica napus ; enoyl-ACP reductase ; fatty acid synthesis ; seed development ; nuclear-encoded chloroplast proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific λgt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1617-4623
    Keywords: Flavonoids ; Antisense genes ; Chalcone synthase ; Chalcone flavanone isomerase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The constitutive expression of an antisense chalcone synthase (CHS) gene in transgenic petunia plants results with high frequency in a reduced flower pigmentation due to a reduction in the CHS mRNA steady-state level in floral tissue. Here we show that this reduction is specific for CHS mRNA; chalcone flavanone isomerase (CHI) and dihydroflavonol reductase (DFR) mRNA steady-state levels are unaffected. However, in white floral tissue a severe reduction in CHI specific activity is found, accompanied by an altered signal for CHI protein on western blots. We find no correlation between the phenotypic effect of the antisense CHS gene and its chromosomal position. For some of the antisense CHS transformants the flower phenotype is highly variable. We demonstrate that pigmentation in these plants can be influenced by gibberellic acid and light, suggesting that the variable flower phenotype is caused by changes in physiological conditions during flower development. The results not only indicate that flower pigmentation in these plants reveals the variable expression of the antisense transgene, but also show that genomic sequences flanking the transgene may render its expression extremely susceptible to physiological conditions.
    Type of Medium: Electronic Resource
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