Abstract:
Ribulose-l,5-bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in photosynthesis. In higher plants, Rubisco is composed of large (LSU) and small subunits (SSU). The LSUs have the active sites, but the function of the SSUs is unclear. The work in this thesis is concerned with analysis of the four closely related SSU genes in the model plant Arabidopsis thaliana. First, to estimate the relative transcript levels of these closely related genes (ATS1A, ATS1B, ATS2B, and ATS3B), a gene expression assay was developed based on the RACE (Rapid Amplification of cDNA Ends) technique. It was found that the relative transcript levels of SSU genes differed significantly between plants grown at different temperatures. In particular, at a low growth temperature (10°C), the total SSU transcript level was dominated by the expression of the ATS1A gene, but the contribution of the ATS]A gene significantly decreased at a high growth temperature (30°C). The relative expression levels of SSU proteins were estimated using two-dimensional electrophoresis (2DE). The comparison of RACE and 2DE analyses demonstrated that the Rubisco SSU protein composition is strongly influenced by the SSU transcript composition at a particular temperature. The observation raised the possibility that an acclimation of Rubisco may be achieved through differential synthesis of SSU proteins, which is in turn determined by differential expression of SSU genes at the transcript level. In order to analyse the function of the different SSU genes further, Arabidopsis SSU mutants were isolated using three different approaches. First, by screening mutant pools generated by insertional mutagenesis, mutations in the SSU genes were identified and null mutants were isolated in the two most strongly expressed genes (ATS1A and ATS3B). Second, by transforming plants with a hairpin construct (RNAi), silencing of the ATS2B gene was achieved in an ATS1A mutant background. In the third, overproduction of individual SSU genes was attempted. The SSU protein compositions in the transformants were changed by transgene expression of individual SSU genes (ATS]A and ATS3B). Under standard greenhouse growth conditions, no obvious phenotype was detected in the SSU mutants. Kinetic constants in the Rubisco carboxylase activity were then estimated in plants that had different SSU compositions. No significant differences were found in the ratio of Km or Vmax between different assay temperatures, which induced the maximal differential SSU expression patterns. At the level of the simple kinetic assays performed here, it appears that the carboxylase activity of Rubisco enzyme is not affected by differential SSU composition. More sophisticated assays may be necessary to discover the role of the SSU in Rubisco.