Analysis of further regulatory effects on the synthesis of 5-aminolevulinic acid by auxiliary factors in Arabidopsis thaliana

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Tetrapyrrole biosynthesis (TPB) in plants consists of more than twenty enzymatic steps and is tightly controlled because of the synthesis of photoreactive intermediates and the different spatio-temporal demands of their end-products chlorophyll and heme. The proposal addresses the control of the rate-limiting synthesis of 5-aminolevulinic acid (ALA) at the beginning of the TPB pathway, which is carried out by the two enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAT). The posttranslational control of ALA synthesis is conceptually based on different mechanisms ensuring the balanced metabolic flow in TPB on the level of activity, stability, oligomerisation and subplastidal compartmentation of the participating enzymes. The objectives are based on the preliminary characterization of TTP1 and TTP3, two novel auxiliary factors of TPB derived from the family of tetratricopeptide-repeat (TPR) proteins, which can most likely be assigned functions in the control of ALA synthesis because of the proven interaction with GluTR. Experiments are proposed to unravel whether both TPR proteins are required for stable association of ALA synthesis enzymes in the chloroplasts, either to promote inactivation of ALA synthesis at the membrane (a hypothesized role for TTP1) or to implement a modified ALA synthesis by feedback-control in the stroma (a proposed role for TTP3). The studies include physical interaction studies between the TPB enzymes and their cooperating regulatory factors as well as the characterization of the single ttp1 or ttp3 mutants and double mutants, which contain a mutation for other known regulatory factors of TPB, such as the FLU protein. Finally, ttp3 and TTP3-overexpressing lines are biochemically analyzed for the additional regulatory connection of ALA synthesis with the initial steps of the porphyrin-synthesis of TPB and TTP3-interaction studies are intended. In general, these studies are expected to gain more knowledge on the complex control of ALA synthesis during day and night-time as well as during changing light intensities.