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.