The regulation and role of plant invertases.

The aim of this thesis was to investigate the regulation and role of invertases in plant carbohydrate metabolism. In the first part of this thesis a molecular approach was adopted and the expression of five invertase genes were examined in different organs and in leaves of different ages in the model plant species Arabidopsis thaliana (L. ). Of the five genes examined two encoded apoplastic invertases (ATßFRUCT 1 and ATßFRUCT 2), two encoded soluble invertases with a probable vacuolar localisation (ATßFRUCT 3 and 4) while the fifth encoded an invertase with an unknown subcellular localisation (ATßFRUCT 5). Generally, Northern hybridisation assays were sufficiently sensitive for the detection of invertase gene expression in A. thaliana, however, in order to examine the expression of rare ATßFRUCT 1 and ATßFRUCT 2 mRNA the reverse transcriptase polymerase chain reaction (RT-PCR) was the method of choice. The development of an RT-PCR internal standard enabled these data to be semi-quantitative in nature. Expression analysis revealed that each of the five invertase genes were differentially regulated in A. thaliana. However, high levels of invertase gene expression were associated with tissues typically considered sinks for carbohydrate. Examination of these tissues also revealed a relationship between invertase activity and the ratio of sucrose to hexoses. Previous work has shown that certain environmental stimuli can influence invertase activity and gene expression. In this thesis the infection of A. thaliana leaves with the biotrophic pathogen Albugo candida resulted in the localised stimulation of cell-wall associated invertase activity. Examination revealed that the majority of this increase was attributable to the stimulation of the host apoplastic invertase gene, ATßFRUCT 1. Furthermore, expression of this gene was also elevated in response to mechanical leaf wounding. The high expression of ATßFRUCT 1 in sink tissues and in response to pathogenesis and wounding suggested that this gene plays an important role in establishing a supply of hexoses to tissues under a wide range of conditions. In the second part of this thesis transgenic tomato plants (prepared by Zeneca Plant Science) with a range of leaf vacuolar invertase activities were examined. There was a linear relationship between vacuolar invertase activity and the amount of leaf hexose. In plants with no detectable leaf vacuolar invertase activity there was an accumulation of sucrose. This suggests that hexoses generated in the leaves of tomato plants are the product of sucrose cycling through the vacuole. The implications of such cycling are discussed in detail