Browsing by Author "Samodien, Mugammad Ebrahim"

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    Analysis of enzymes involved in starch phosphate metabolism
    (Stellenbosch : University of Stellenbosch, 2009-12) Samodien, Mugammad Ebrahim; Lloyd, James Richard; Kossmann, J. M.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)
    ENGLISH ABSTRACT: This project examined the role of proteins in starch phosphate metabolism. The first part was aimed at the functional characterization of the SEX4, LSF1 and LSF2 genes in both plants and bacteria. Constructs were produced to allow for expression of the three proteins in E. coli with the SEX4 and LSF2 proteins being successfully purified and used to produce antibodies. Immunoblot analysis indicated that the antibodies recognised the repective proteins in extracts, but it was not clear if they actually recognised the proteins or the GST tags they were fused to. Virus induced gene silencing constructs were also produced to allow repression of these three genes in Nicotiana benthamiana. This resulted in a starch excess phenotype being observed in the leaves of silenced plants which is consistent with the known or presumed roles for the genes. The antibodies produced were not specific enough to confirm that the respective protein were actually repressed, but it is likely that this was the case as plants infiltrated at the same time with a VIGS vector designed to repress phytoene desaturase exhibited a chlorophyll bleaching phenotype. These data confirm that SEX4 and LSF1 probable play the same role in N. benthamiana as in Arabidopsis, and provide evidence that LSF2 is also necessary for starch degradation. It was also attempted to characterise these proteins with respect to their substrate utilization by setting up a glyco-array experiment. Various potato starches from genetically modified plants were subjected to hydrolytic attack by starch degrading enzymes and fractionated by anion exchange chromatography to produce a multitude of glucans. These will be spotted onto glass filters and probed with the purified proteins to see if they bind to specific starch breakdown products preferentially. iv The project also involved investigating the effect the SEX4 protein has on E. coli glycogen contents. SEX4 was expressed in wild type and glgX mutant E. coli strains as it has been shown that this stops glycogen accumulation in the wild type, but not the glgX mutant. The cells were grown in liquid culture and glycogen contents measured. In liquid cultures SEX4 had no effect on glycogen contents in the wild type, possible because of problems with plasmid stability in the strain used. This final part of the project investigated the effect that a gwd mutation has on carbohydrate metabolism in leaves and fruits of the Micro-tom tomato cultivar. Starch and soluble sugar contents were measured in leaves and ripening fruits. A starch excess phenotype was found in the leaves, but no change in starch contents was determined in either the placenta or pericarp of the fruit. Soluble sugar contents were reduced in the fruit tissues, although the reason for this in unclear.
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    The analysis of starch degradation in Solanaceae species
    (Stellenbosch : Stellenbosch University, 2014-04) Samodien, Mugammad Ebrahim; Lloyd, James Richard; Kossmann, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics ​and Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: This project involved the analysis of genes in Solanaceae species that have previously been shown to be involved in the phosphorylation of starch or its subsequent dephosphorylation. Both these processes are essential for normal starch mobilization. A tomato conditional mutant lacking the starch phosphorylating enzyme glucan water dikinase was analyzed. It is known that starch accumulates transiently in tomato fruit and is degraded throughout the ripening process. The study aimed to determine the effect of inhibited starch degradation on fruit development. Unfortunately no effect on starch mobilisation was found in the fruit of the mutant. Immunoblot analysis revealed expression of Glucan Water Dikinase (GWD) within the fruit of the tomato mutant indicating that the conditionality of the mutation was compromised. The second set of experiments analyzed the roles of Starch Excess4 (SEX4), Like Sex Four-1 and Like Sex Four-2 (LSF1 and LSF2) in starch degradation in potato and Nicotiana benthamiana. These enzymes have, thus far, only been studied in Arabidopsis, with the proposed role for SEX4 and LSF2 being that they are involved in dephosphorylation of the C-6 and C-3 positions of starch breakdown products. The role of LSF1 is unclear, although it is not thought to be a phosphatase. SEX4, LSF1 and LSF2 were repressed individually while the expression of SEX4 and LSF2 were also inhibited simultaneously. Using a transient repression system in N. benthamiana it was shown that all of the genes play a role in leaf starch degradation. The SEX4 and LSF2 enzymes were shown to influence the proportion of phosphate located on the starch which contained an altered ratio of C-3/C-6 phosphate. Stably transformed potato plants were produced where SEX4 and LSF2 were successfully repressed in potato leaves and tubers. Although AtLSF2 had been shown not to be essential for normal starch degradation on its own, in potato plants when LSF2 was repressed, the plants developed a starch-excess phenotype. Taken together with the N. benthamiana data this indicates that LSF2 plays a bigger role in leaf starch degradation in Solanaceae than in Arabidopsis. The ratio of C-3/C-6 phosphate was also altered in tuber starch from some of the silenced plants. Starch from SEX4 repressed potato plants contained increased amounts of glucose-6-phosphate and increased glucose-3-phosphate in the tuber when compared to the WT. An increase in the proportion of C-6 or C-3 phosphate is not surprising with SEX4 being characterized as a phosphatase specific for C-6 position and LSF2 for the C-3 position in Arabidopsis, however the combined increase in C-3 and C-6 amounts in StSEX4 silenced plants is interesting. The differences seen in the phosphate alteration in both N. benthamiana leaves and potato tubers indicates that in Solanaceae species these proteins may have a slightly altered specificity when compared with Arabidopsis, although they are undoubtedly involved in starch degradation. The effect of silencing SEX4 or LSF2 on cold-induced sweetening was also investigated, with no effect being found. This may be because of functional redundancy between the proteins and a better approach in terms of blocking cold sweetening would be to simultaneously repress SEX4 and LSF2. Overall, these enzymes seem to play similar roles in leaves of Solanum species as has been described in Arabidopsis. The starch from the engineered plants did have an altered phosphate ratio and further analysis is needed to determine if this leads to improved or additional functionality.