10 research outputs found
Downregulation of pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexose-phosphate levels
Analyses of transgenic sugarcane clones with 45–95% reduced cytosolic pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypical change, but significant changes were evident in in vivo metabolite levels and fluxes during internode development. In three independent transgenic lines, sucrose concentrations increased between three- and sixfold in immature internodes, compared to the levels in the wildtype control. There was an eightfold increase in the hexose-phosphate:triose-phosphate ratio in immature internodes, a significant restriction in the triose phosphate to hexose phosphate cycle and significant increase in sucrose cycling as monitored by 13C nuclear magnetic resonance. This suggests that an increase in the hexose-phosphate concentrations resulting from a restriction in the conversion of hexose phosphates to triose phosphates drive sucrose synthesis in the young internodes. These effects became less pronounced as the tissue matured. Decreased expression of PFP also resulted in an increase of the ATP/ADP and UTP/UDP ratios, and an increase of the total uridine nucleotide and, at a later stage, the total adenine nucleotide pool, revealing strong interactions between PPi metabolism and general energy metabolism. Finally, decreased PFP leads to a reduction of PPi levels in older internodes indicating that in these developmental stages PFP acts in the gluconeogenic direction. The lowered PPi levels might also contribute to the absence of increases in sucrose contents in the more mature tissues of transgenic sugarcane with reduced PFP activity
Manipulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in sugarcane
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006.The main aim of the work presented in this thesis was to elucidate the apparent role of
pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation
in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the
sucrose content and positively to the water-insoluble component across varieties which
differ in their capacities to accumulate sucrose. This apparent well defined and important
role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general
literature as well as the results of various transgenic studies where neither the downregulation
nor the over-expression of PFP activity had a major influence on the phenotype
of transgenic potato and tobacco plants. Based on this it was therefore thought that either
the kinetic properties of sugarcane PFP is significantly different than that of other plant
PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch
accumulating tissues.
In the first part of the study sugarcane PFP was therefore purified and its molecular and
kinetic properties were determined. It consisted of two subunits which aggregated in
dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the
glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for
all the substrates were comparable to that of other plant PFPs. The conclusion was therefore
that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from
that of other plant PFPs.
The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is
to alter its levels in the same genetic background through genetic engineering. This was
therefore the second focus of this study. PFP activity was successfully down-regulated in
sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field
conditions and sucrose concentrations in their immature internodes were significantly
increased. PFP activity was inversely correlated with sucrose content in the immature internodes of the transgenic lines. Both the immature and mature internodes of the
transgenic plants had significantly higher fibre contents.
This study suggests that PFP plays a significant role in glycolytic carbon flux in immature,
metabolically active sugarcane internodal tissues. The data presented here confirm that PFP
can indeed have an influence on the rate of glycolysis and carbon partitioning in these
tissues. It also implies that there are no differences between the functions of PFP in starch
and sucrose storing tissues and it supports the hypothesis that PFP provides additional
glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue.
This work will serve as a basis to refine future genetic manipulation strategies and could
make a valuable contribution to the productivity of South African sugarcane varieties
Metabolic Changes Associated with the Sink-Source Transition During Sprouting of the Axillary Buds on the Sugarcane Culm
Sucrose, glucose and fructose concentrations, and sucrolytic enzyme activities were measured in the developing shoots and internodes of sprouting sugarcane setts (Saccharum spp, variety N19). The most striking change during the sink-source transition of the internode and germination of the axillary bud is a more than five-fold induction of cell wall invertase in the germinating bud. In contrast, soluble acid invertase is the main sucrose hydrolytic activity induced in the internodal tissue. A cycle of breakdown and synthesis of sucrose was evident in both the internodes and the shoots. During shoot establishment, the sucrose content decreased and the hexose content increased in the internodal tissues while both sucrose and hexoses continuously accumulated in the shoots. Over the sprouting period internode, dry mass was reduced by 25 and 30\ua0% in plants incubated in a dark/light cycle or total darkness, respectively. Sucrose accounted for 90\ua0% of the dry mass loss. The most significant changes in SuSy activity are in the synthesis direction in the shoots resulting in a decrease in the breakdown/synthesis ratio. In contrast the SuSy activity in the internodal tissue decrease and more so in the synthesis activity resulting in an increase in the breakdown to synthesis ratio
Plant-made therapeutics: An emerging platform in South Africa
The field of plant-made therapeutics in South Africa is well established in the form of exploitation of the country's considerable natural plant diversity, both in the use of native plants in traditional herbal medicines over many centuries, and in the more modern extraction of pharmacologically-active compounds from plants, including those known to traditional healers. In recent years, this has been added to by the use of plants for the stable or transient expression of pharmaceutically-important compounds, largely protein-based biologics and vaccines. South Africa has a well-developed plant biotechnology community, as well as a comprehensive legislative framework for the regulation of the exploitation of local botanic resources, and of genetically-modified organisms. The review explores the investigation of both conventional and recombinant plants for pharmaceutical use in South Africa, as well as describing the relevant legislative and regulatory frameworks. Potential opportunities for national projects, as well as factors limiting biopharming in South Africa are discussed. © 2011