Browsing Doctoral Degrees (Genetics) by Author "Adegbaju, Muyiwa Seyi"
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- ItemAnalysis of interactions between glucan, water dikinase and either isoamylase or starch branching enzymes in detrmination of starch structure(Stellenbosch : Stellenbosch University, 2022-12) Adegbaju, Muyiwa Seyi; Lloyd, James Richard; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is the main form in which carbon is stored in plants and it is used in a wide array of applications, from its role as a source of calories in human diets, to livestock feed and as an industrial feedstock. It is composed of two types of glucan polymer, amylose which is com- posed mainly of α-1,4 linked chains and highly branched amylopectin which contains α1,4 chains which are connected together with α1,6 branchpoints. Synthesis of the starch polymer involves co-ordinated activities of starch synthases, starch branching enzymes (SBE) and de- branching enzymes (DBE). In addition, the enzyme glucan, water dikinase (GWD) can intro- duce phosphate into amylopectin. The critical roles these enzymes play during starch metabo- lism have been investigated by knockout or silencing of genes encoding them in some plants, mainly Arabidopsis thaliana. Interactions between some of these enzymes have also been demonstrated, but there are still many questions about how these interactions occur and how they influence starch metabolism. In this study two sets of experiments were performed. The first involved analyses of potato (Solanum tuberosum L.) plants where the expression of SBEI, SBEII and/or Glucan, Water Dikinase 1 (GWD1) were repressed using RNAi technology. Individual or joint repression of the two SBE isoforms resulted in a significant increase in starch phosphate, whereas repression of GWD1 led to synthesis of low phosphate starch. Starch phosphate decreased in SBEI/GWD1 and SBEII/GWD1 lines but not to the levels found in lines where only GWD1 was repressed. The apparent amylose content increased in starch from SBEI/GWD1 lines was greater than either the SBEI or GWD1 lines. These alterations in starch composition influenced its granule morphology, swelling power and freeze-thaw stability. Silencing of GWD1 reduced starch deg- radation in cold-stored tubers, but this was not the case in lines repressed in either starch branching enzyme. The second set of experiments examined polyglucan metabolism in potato lines repressed in isoamylase 2 (ISA2) and/or GWD1. Transgenic potato lines were produced by RNAi gene silencing and tuber starch metabolism analysed. The water-soluble glucan content in ISA2 and ISA2/GWD1 lines tuber was higher than GWD1 lines. Analysis of starch structure indicated that there was an increase in the apparent amylose content of starches from GWD1 lines whereas it was unchanged ISA2/GWD1 lines. There was a reduction in the starch phosphate in both GWD1 and ISA2/GWD1 lines, and this reduction was greatest in the GWD1 lines. The ISA2 lines contained a proliferation of small starch granules, but this was not observed in ISA2/GWD1 lines which indicates that ISA2 and GWD1 interact during granule initiation. The effect of both enzymes on cold-induced sweetening (CIS) was also investigated. As expected, CIS was reduced in lines where GWD1 was silenced. Interestingly CIS was also inhibited in the ISA2 lines indicating that the pathway of starch degradation may differ between cold-stored potato tubers and Arabidopsis leaves.