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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The role of fructose-2,6-bisphosphate (Fru-2,6-P2) in regulation of carbon metabolism was investigated in transgenic potato plants (Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P2 was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic 14CO2 metabolism, showed that in plant lines with reduced Fru-2,6-P2 level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P2 only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-14C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-14C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P2 is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P2 on tuber metabolism was limited.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0983
    Keywords: Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-0983
    Keywords: Key words Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-5028
    Keywords: carbohydrate metabolism ; fructose-2,6-bisphosphate ; fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase ; potato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated cDNA clones encoding the regulatory enzyme fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from a potato (Solanum tuberosum) leaf cDNA library. All clones represented transcripts of the same gene (F2KP1). Functionality of the encoded protein was verified by expression of the active enzyme in Escherichia coli. The expressed enzyme had both kinase activity which forms fructose-2,6-bisphosphate from fructose-6-phosphate and ATP, and phosphatase activity which degrade fructose-2,6-bisphosphate. The recombinant potato enzyme was radiolabelled by [2-32P]fructose-2,6-bisphosphate verifying conservation of the phosphatase catalytic mechanism which involves a phospho-protein intermediate. The deduced amino acid sequence corresponding to the catalytic core for F2KP1 is homologous to the fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase isolated from animals and yeast, with conservation of amino acids involved in substrate binding and catalytic mechanisms. The sequence for F2KP1 also includes a 102 amino acids long NH2-terminal with no homology to any previously identified enzymes. This NH2 terminal may be even longer since an upstream stop codon has not yet been identified. Northern blot analysis of potato showed that the F2KP1 transcript is present in several tissues including source leaves, sink leaves and flowers, whereas the transcripts were not detectable in developing tubers. Southern blot analysis of Solanum phureja suggest there to be only one copy of the gene.
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