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Title: Investigation of the role of disproportionating enzyme in starch metabolism by isolation and characterisation of a mutant of Arabidopsis thaliana (L.)
Author: Critchley, Joanna Harriet
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Mutants deficient in various enzymes have already contributed much to current understanding of starch metabolism. Arabidopsis thaliana now offers the potential to isolate specific stable null mutants in target genes which should prove invaluable in determining how enzymes work together in transitory starch metabolism. D-enzyme, disproportionating enzyme, or 4-a-glucanotransferase (EC catalyses glucan transfer in vitro from one 1,4-a-D-glucan molecule to another, to itself to form cyclic molecules, or to glucose. In vitro, the preferred substrate of the enzyme is malto-oligosaccharides (MOS). A number of different roles for D-enzyme have been suggested in the breakdown and synthesis of starch. In Arabidopsis D-enzyme was found to be expressed in all plant organs investigated, and at all times over a 24 hour time course. This study uses a reverse-genetics approach to isolate a mutant of Arabidopsis lacking D-enzyme. cDNA and genomic sequences encoding D-enzyme were isolated from Arabidopsis libraries. The sequence was used to design primers for PCR screening of DNA from plants containing random T-DNA insertions. 19 000 plants were screened and one plant line was found in which the D-enzyme gene was disrupted by a T-DNA insert. Western blot analysis and enzyme assays confirmed that the homozygous mutant lacked D-enzyme protein and activity. Mutant plants grew 30% more slowly than wild-type plants and leaves had a higher starch content than the wild-type throughout the diurnal cycle. At the end of the night an appreciable amount of starch remained in the leaves of the mutant that had been degraded in the wild-type leaves. Mutant plants also accumulated high levels of MOS from the start of the dark period, indicating that D-enzyme is responsible for metabolising MOS produced during the nocturnal breakdown of starch. During the day, the mutant was shown to have normal levels of MOS, indicating that, in higher plants, MOS are probably not a substrate for D-enzyme during the synthesis of starch.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available