The manipulation of fructose 2,6-bisphosphate levels in sugarcane

Thesis

Hiten, Nicholas Fletcher
Abstract

Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 2.7.1.105) and fructose 2,6-bisphosphatase (FBPase2, EC 3.1.3.11) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.

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