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Transketolase is a TPP dependent enzyme that affects the availability of intermediates in both the Calvin cycle and non-oxidative pentose phosphate pathway. Previous studies have indicated that changes to the activity level of transketolase can limit growth and development as well as the production of isoprenoids, starch, amino acids and thiamine. The overall aim of this project was to further advance the understanding of the mechanism linking increased TK activity and thiamine metabolism. Nicotiana tabacum mutants with increased total transketolase activity ~ 2 to 2.5 fold higher than WT plants were shown to have a reduced growth and chlorotic phenotype. In seedlings, these phenotypes were attributed to a reduction in seed thiamine content. Imbibition of TKox seeds in a thiamine solution produced plants that were comparable to WT plants. However, the chlorotic but not growth phenotype was found to return unless the plants underwent irrigation with a thiamine solution indicating that TKox plants are unable to produce sufficient quantities of thiamine to meet demand. Furthermore, the application of deoxy-xylulose-5-phosphate was also found to be able to partially complement the phenotype suggesting that flux from the C3 cycle into the non-mevalonate pathway is being affected. Analysis of thiamine and TPP levels demonstrated that TKox plants were deficient in thiamine but not TPP in the majority of cases. In plants that had begun to flower, TKox lines had reduced thiamine and TPP levels in the 20th fully open leaf compared to the same leaf in WT plants. Furthermore, sampling of leaf tissue from both WT and TKox seedlings at the same developmental stage indicated that high levels of TK protein may lead to the accumulation of TPP in these areas causing a reduction in the levels of thiamine and TPP in the rest of the plant thereby limiting growth and development. |
