Investigation of the regulation of carbohydrate metabolism in Arabidopsis thaliana using a genetic approach

Abstract

The regulation of carbohydrate metabolism in Arabidopsis thaliana was investigated using a genetic approach. A new class of carbohydrate insensitive mutant (cai) was characterised in order to gain insight into the control of carbohydrate metabolism. Wild type seedlings germinated on media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons did not expand and accumulated anthocyanins. After 1 week growth was arrested. The internal carbohydrate content increased accompanied by repression of photosynthetic genes and induction of chs gene expression, cai mutants germinated on agar media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons expanded and greened. After initial characterisation of a number of the mutants, two were selected for further analysis. When germinated on a range of different carbon; nitrogen ratios cai 10 and cai 28 displayed a reduced sensitivity to the high carbohydrate and low nitrogen conditions, cai 10 also displayed a mannose insensitive (mig) phenotype compared to the post-germinative growth of wild type which was arrested by mannose. This growth arrest in the wild type on mannose correlates with phosphate sequestration, cai 10 metabolises mannose at a different rate and accumulates less hexose phosphate than the wild type when germinated on mannose, thus indicating that the mannose insensitive phenotype may be a consequence of a disruption in metabolism. Overexpression of Arabidopsis hexokinase 1 in cai 10 did not complement the cai 10 phenotype. In contrast to previous results by Jang et al., (1997), who found that plants overexpressing hexokinase were hypersensitive to sugars, our results indicate that they are less sensitive than wild type. This is not in agreement with the proposed model of hexokinase as a sugar sensor (Jang et al., 1997). Seeds of the hexokinase overexpressors germinated rapidly (within 18-20 h). The seeds also contained elevated levels of some amino acids, smaller lipid bodies and less lipid than the wild type. It is proposed that hexokinase overexpression increases glucose-6-phosphate concentration which activates phosphoenolpyravate carboxylase (PEPCase) and in so doing diverts carbon from lipid biosynthesis to amino acid synthesis