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  • 1
    ISSN: 1573-5028
    Keywords: Cell division ; gene expression ; Nicotiana sylvestris ; protoplast ; stress ; ubiquitin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5- mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; extensin ; hydroxyproline-rich cell wall protein ; Nicotiana sylvestris ; protoplasts ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5028
    Keywords: HMG-CoA reductase ; isoprenoid biosynthesis ; Nicotiana sylvestris ; protoplast ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA library from freshly isolated mesophyll protoplasts of Nicotiana sylvestris was differentially screened using cDNAs from leaves, leaf strips submitted to the same stress as protoplasts during the isolation procedure, and cell suspension cultures. One of the selected clones (6P2) was found to encode a putative polypeptide highly homologous to previously characterized 3-hydroxy-3-methylglutaryl coenzyme A reductases. The C-terminal region of the polypeptide was highly conserved whereas its N-terminal region including the trans-membrane domains and the linker was more variable. Apart from protoplasts, the 6P2 gene was found to be expressed in apexes, anthers, roots, and in stressed leaf strips after 24h of culture, during the hypersensitive reaction to viral infection and after HgCl2 treatment. This pattern of expression is consistent with a role in plant defence mechanisms.
    Type of Medium: Electronic Resource
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