Browsing by Author "Jacobs, Ingrid"

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    Analysis of aspects of the starch metabolic pathway in lower plants
    (Stellenbosch : Stellenbosch University, 2018-12) Jacobs, Ingrid; Lloyd, James Richard; Stellenbosch University. Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is an important plant storage polysaccharide that has been demonstrated to have a major influence on plant growth. Transitory starch is synthesized in the leaves of plants during the day as a product of photosynthesis and degraded at night to allow continued carbon allocation for growth and cellular processes. It is also produced and stored for longer periods of time in non-photosynthetic organs such as stems, tubers and seeds. The study of starch is important for several reasons – not only is it a vital part of the human diet, it is also utilised in many non-food applications such as the paper, textile, oil and pharmaceutical industries. The pathway of starch metabolism in higher plants has been studied for decades and of late, Arabidopsis has become the workhorse plant for many starch researchers due to the plethora of insertion mutants that are readily available. However, the predominant use of Arabidopsis as a model system has led to a narrow understanding of starch metabolism restricted to that of synthesis and degradation of leaf starch. There are ongoing attempts to translate the knowledge gleaned from Arabidopsis into studies on the storage organs of crop plants (e.g. rice and maize endosperm, potato tubers), as well as starch metabolism in lower plants (e.g. algae and mosses) to aid in elucidating the evolutionary development of starch metabolism in land plants. This study investigated two aspects of starch metabolism in lower plants to determine whether the pathway of starch metabolism observed in higher plants is conserved. Firstly, a previously uncharacterised starch synthase from the red alga Chondrus crispus was examined due to the reported differences in substrate preference between red algal and green plant starch synthases, and the deviation in compartmentalisation of starch synthesis and storage in members of the red algae. The C. crispus starch synthase was analysed by means of multiple sequence alignment, site-directed mutagenesis and recombinant protein expression and purification. Features unique to red algal starch synthases were identified, including a C-terminal glycogen binding domain and sequence variations in important residues involved with substrate binding. During recombinant expression, the C. crispus protein was insoluble and accumulated in inclusion bodies. Attempts to recover active protein through optimisation of expression, the use of alternative expression systems and protein refolding were unsuccessful and biochemical characterisation of the starch synthase could not be performed. Secondly, four putative orthologues of the Arabidopsis maltose excess (MEX) transporter were identified in the moss Physcomitrella patens and their functions examined through the generation of knockout mutant lines and complementation of Escherichia coli mutants defective for sugar transporters. Knockout mutants were successfully generated for the P. patens MEX1a gene, while complementation studies failed to produce active protein. Expression profiling in wild type P. patens suggest that the four PpMEX genes are differentially expressed depending on the developmental stage of the culture and may have specialised functions in various growth structures.