Investigating the role of pyrophosphate fructose 6-phosphate 1-phosphotransferase in phloem loading

[摘要] The main aim of the work presented in this thesis was to further our understanding of therole of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, byspecifically investigating its potential contribution to phloem metabolism. PFP activity insugarcane internodal tissue is inversely correlated to sucrose content across varieties thatdiffer in their sucrose accumulation abilities. This apparent correlation is in contrast toprevious studies that suggest PFP plays an insignificant role in metabolism.In the first part of this study an immunological characterisation of the two subunits ofsugarcane PFP was conducted to establish whether it differ significantly from other plantspecies in terms of size and distribution. Both the alpha and beta subunit appears to beapproximately sixty kilo Daltons in size and uniform in their relative distribution to eachother in the various plant organs of sugarcane. Although the observed alpha subunit size isless than that predicted this could be explained at the hand of post translationalmodification, in essence the sugarcane PFP subunits appear similar than that described forother plants especially that of tobacco which was employed as a model system later on inthis study.The only direct way to investigate PFP's contribution to phloem metabolism is to alter itsactivity by recombinant DNA technologies. Therefore, in the second part of the studytransformation systems were designed for both the constitutive and phloem specific downandup-regulation of PFP activity. For the down-regulation of activity a post transcriptionalgene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencingsystem, was employed. A partial sequence of the PFP-beta subunit was isolated and used invector construction. For the over-expression the Giardia lamblia PFP gene was used. Themodel plant tobacco was employed to investigate PFP's effect on phloem metabolism andtransport of assimilate. Transgene insertion was accomplished by means of Agobacteriummediated transformation and tissue specific manipulation of PFP activity was confirmed byin situ activity staining.

[发布机构] Stellenbosch University