Browsing by Author "Wiese, Anna Johanna"
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- ItemCharacterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens.(Stellenbosch : Stellenbosch University, 2020-12) Wiese, Anna Johanna; Lloyd, James Richard; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.ENGLISH ABSTRACT: Plant sugars have dual functionality, in that they play a role in primary metabolism and partake in signal transduction pathways. As it relates to their signaling function, they relay information to the nucleus regarding energy status, allowing the plant to adapt accordingly. Most of what is known about these functions of sugars have come from research in vascular plants, leaving aspects thereof unaddressed in non-vascular plants. Certain bryophytes (non-vascular plants) have been developed over into model plants, with Physcomitrella patens representing one such model. This plant has become popular in studies of evolutionary development and non-vascular plant biology. In this study, I characterized two gene families in P. patens, namely sucrose synthases (SUS) and trehalose 6-phosphate synthases (TPS), whose homologs in higher plants are implicated in sugar metabolism and signaling. Sucrose, the end-product of photosynthesis, is central to primary carbon metabolism. Its synthesis and degradation are tightly controlled to balance out supply and demand throughout the plant. Sucrose synthases are implicated in phloem loading and sink strength in vascular plants, where its cleavage products can enter primary metabolism or be used in the synthesis of complex carbohydrates. Little is known about SUS function in non-vascular plants, and in this study, I report the characterization of four putative SUS homologs in P. patens. Phylogenetic classification of land plant SUS sequences revealed the existence of 5 clades, one of which contained only bryophyte-sequences including all those from P. patens. Analysis of the amino acid sequences revealed that residues involved in SUS regulation in higher plants were conserved in PpSUS proteins. I was able to demonstrate SUS activity in crude protein extracts, however, detailed kinetic characterization was hindered by protein expression in E. coli. Localization studies revealed that all PpSUS proteins were cytosolic, while expression analyses indicated that PpSUS genes have overlapping and unique expression patterns. Another sugar which is implicated in signaling is trehalose 6-phosphate (Tre6P), an intermediate in the trehalose biosynthesis pathway. Levels of this sugar phosphate change in parallel to that of sucrose, with researchers proposing that it plays a role in communicating sucrose availability. The second part of this study focussed on the proteins involved in Tre6P synthesis, namely trehalose 6-phosphate synthases (TPSs), which are divided into two classes, with class I proteins containing catalytically active polypeptides (Leyman et al., 2001; Lunn, 2007). Very little is known about the class II proteins, and in this study, I characterized members of this class in P. patens. Physcomitrella contains six TPS genes in its genome, four of which encode class II proteins. Phylogenetic classification differentiated TPS sequences from land plants into 2 clades (I and II) consisting of 7 sub-clades (IA-B and IIA-E), suggesting the existence of 1 ancestral TPS gene. Functional complementation revealed weak TPS catalytic activity for one of the class II TPS proteins, a first for any plant class II protein studied to date. Subcellular localization experiments conducted on three of the class II proteins revealed that they were cytosolic, while yeast two hybrid analyses indicated that these proteins do not form complexes with each other or the class I proteins. Finally, expression analyses indicated that class II genes have overlapping expression patterns. This study provides novel insights into the evolution of SUS and TPS genes in P. patens and, will serve as a platform for the design of future experiments related to these gene families in non-vascular plants.
- ItemComparative analyses of primary carbon metabolism in parasitic plant species(Stellenbosch : Stellenbosch University, 2013-12) Wiese, Anna Johanna; Lloyd, James Richard; Van der Merwe, Margaretha; Stellenbosch University. Faculty of Agricultural. Dept. of Genetics.ENGLISH ABSTRACT: Most terrestrial plants make use of beneficial symbiotic associations to obtain nutrients (eg. nitrogen (N) and phosphorous (P)) from fungi in exchange for photoautotrophic carbon. However, plant parasitism (defined here as the ability of certain plants to parasitize other living material) has evolved in the plant kingdom and such plants obtain some, or all, of their nutritional needs from a host, which is severely negatively impacted by the parasite. While the physiological adaptations are well studied, the underlying molecular and biochemical mechanisms of plant parasitism remain largely unknown. As a first approach, a biochemical blueprint of primary metabolites present within parasitic plant species was constructed. The metabolomes of nineteen parasitic plants, ranging from hemi- and holoparasitism to mycoheterotrophism, were profiled via gas chromatography mass spectrometry (GC MS) based technology and targeted spectrophotometric assays. Based on these analyses, three important observations were made. First, parasitic plants were severely carbon deprived, despite being successful in colonizing and exploiting their hosts. Second, the levels of organic acids participating in mitochondrial respiration decreased and certain amino acids and soluble protein content increased. This suggests that parasitic plants utilize alternative respiratory substrates to compensate for a limitation in carbon supply. Third, although characterized by reduced carbohydrate pools, minor sugars normally not associated with plant metabolism, dominated the soluble sugar pool. The presence and significance of one of these sugars, namely turanose (α-D-glucopyranosyl-(1→3)-α-D-fructofuranose), was further investigated. Turanose biosynthetic reactions could be demonstrated in Orobanche minor extracts. Protein purification and mass spectrometry identification suggested that turanose biosynthesis occurred uniquely in parasitic plants. Future work will elucidate the functional significance of turanose metabolism in plant parasitism. Taken together, this study significantly contributes to our understanding of plant parasitism through development of metabolic signatures associated with distinct parasitic classes. These biochemical profiles highlighted several important strategies and alternative metabolic pathways that are either expressed or constitutively activated during parasitism. This knowledge broadens the scope of using parasitic plants in several biotechnological applications or as a novel research tool to address fundamental questions in plant science.