title: The regulation of phosphoenolpyruvate carboxylase in higher plants
Creators: name: Hartwell, James
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Plant sciences

description: The aim of the work in this thesis was to investigate the requirement for protein synthesis in the induction of phosphoenolpyruvate carboxylase (PEPc) kinase in higher plants. A novel assay was developed in which isolated plant RNA was translated in vitro using a rabbit reticulocyte lysate system and the translation products were assayed directly for PEPc kinase activity. A series of experiments were performed which demonstrate that the assay provides a valid estimate of the amount of PEPc kinase translatable mRNA in any sample of RNA. The extent of labelling of exogenous PEPc in the assay is proportional to the volume of translation products used, the amount of RNA translated and (after a short lag) the duration of the in vitro translation. Inclusion of EGTA in the kinase assays of translation products demonstrated that the kinase activity produced was Ca2+-independent, like the activity detected in plant extracts. Size fractionation of a sample of mRNA revealed that PEPc kinase mRNA has a length of between 0.9 and 1.3 Kb which corresponds well with the predicted size calculated from the molecular weight of partially purified PEPc kinase polypeptides. In the leaves of the CAM plant Bryophyllum (Kalanchoe) fedtschenkoi, in normal diurnal conditions, kinase mRNA was high at night and below the limits of detection during the day. In constant environmental conditions (continuous darkness, CO2-free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. Pharmacological agents were used to dissect the signal transduction cascade that mediates circadian regulation of PEPc kinase mRNA levels and PEPc kinase activity. The nocturnal appearance of kinase mRNA and activity was blocked by a number of protein and RNA synthesis inhibitors which act on different components of the transcription and translation machinery. In addition to transcription and translation, the circadian appearance of PEPc kinase mRNA and activity requires protein dephosphorylation and a calcium/calmodulin interaction. The physiological relevance of changes in PEPc kinase mRNA and activity to CAM with respect to CO2 assimilation and total leaf malate content was investigated using intact plants of Kalanchoe daigremontiana. The leaf malate content was manipulated by placing leaves in an atmosphere of pure nitrogen during the night for various lengths of time to prevent CO2 fixation and respiration. The effects of blocking nocturnal CO2- fixation and malate accumulation on PEPc kinase mRNA and activity levels were examined with a view to determining whether the metabolite status of the leaf could influence the circadian control of the allosteric properties of PEPc. Changes in CO2 fixation and PEPc kinase activity reflected those in kinase mRNA except in leaves transferred from nitrogen to ambient air at the start of the light period. In these leaves, kinase activity fell even though kinase mRNA rose, probably due to a rapid increase in cytosolic malate. The highest rates of CO2 fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. A temperature increase from 19°C to 27°C led to a rapid reduction in kinase mRNA and activity. However, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These results demonstrate that the metabolite status of the leaf and in particular the leaf malate status can override the circadian clock control of PEPc kinase mRNA and activity.