Investigating the role of pyrophosphate fructose 6-phosphate 1-phosphotransferase in phloem loading
Thesis (MSc (Genetics. Plant Biotechnology)) --Stellenbosch University, 2008.
The main aim of the work presented in this thesis was to further our understanding of the role of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, by specifically investigating its potential contribution to phloem metabolism. PFP activity in sugarcane internodal tissue is inversely correlated to sucrose content across varieties that differ in their sucrose accumulation abilities. This apparent correlation is in contrast to previous studies that suggest PFP plays an insignificant role in metabolism. In the first part of this study an immunological characterisation of the two subunits of sugarcane PFP was conducted to establish whether it differ significantly from other plant species in terms of size and distribution. Both the alpha and beta subunit appears to be approximately sixty kilo Daltons in size and uniform in their relative distribution to each other in the various plant organs of sugarcane. Although the observed alpha subunit size is less than that predicted this could be explained at the hand of post translational modification, in essence the sugarcane PFP subunits appear similar than that described for other plants especially that of tobacco which was employed as a model system later on in this study. The only direct way to investigate PFP’s contribution to phloem metabolism is to alter its activity by recombinant DNA technologies. Therefore, in the second part of the study transformation systems were designed for both the constitutive and phloem specific downand up-regulation of PFP activity. For the down-regulation of activity a post transcriptional gene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencing system, was employed. A partial sequence of the PFP-beta subunit was isolated and used in vector construction. For the over-expression the Giardia lamblia PFP gene was used. The model plant tobacco was employed to investigate PFP’s effect on phloem metabolism and transport of assimilate. Transgene insertion was accomplished by means of Agobacterium mediated transformation and tissue specific manipulation of PFP activity was confirmed by in situ activity staining.