On the functional roles of the xylulose 5-phosphate/phosphate translocator and related transporters in primary and secondary metabolism of Arabidopsis thaliana
Final Report Abstract
The four different phosphate translocators (PT) of the inner envelope membrane of plastids fulfil numerous tasks in the controlled exchange of phosphorylated metabolic intermediates and inorganic phosphate (Pi) between cytosol and stroma. (1) The triose phosphate PT (TPT) in leaves exports TP from CO2 assimilation as a substrate for sucrose synthesis from the stroma to the cytosol. (2) The glucose 6-phosphate PT (GPT), on the other hand, supplies the stroma of non-green plastids with Glc6P as a substrate for the synthesis of storage starch and/or for the oxidative pentose phosphate pathway (OPPP), during which redox power in the form of NADPH and precursors for anabolism are formed. (3) The phosphoenolpyruvate (PEP) PT (PPT) is expressed in green and non-green tissues and supplies the stroma with PEP, which together with erythrose 4-phosphate serves as a precursor for the shikimate pathway to synthesise aromatic amino acids, from which a large number of important plant constituents are subsequently derived. (4) The role of the xylulose 5-phosphate PT (XPT), which links the plastidic to the extraplastidic OPPP, is poorly understood. XPT mutants in A. thaliana do not show a phenotype distinguishable from the wild type. However, double mutants in which the function of XPT and TPT are simultaneously inactivated show a clear impairment of growth and altered photosynthetic properties similar to those of the adg1/tpt double mutant. There, both the export of photoassimilates from the chloroplast and the formation of transitory starch are inhibited. Based on the findings of the first funding period, the understanding of the role of the XPT, especially in combination with the TPT, was intended to be deepened in the following three subprojects. (1) The role of XPT under stress conditions, (2) detailed analysis of the phenotype of the xpt/tpt double mutant, (3) alternative pathways for PEP import into plastids. Ad 1 The XPT is involved in the recovery of pentose phosphates from the extraplastidic branch of the OPPP. Pathogens as well as salt and osmotic stress increase the flux through this branch of the OPPP and lead to increased formation of NADPH and reactive oxygen species. Stress conditions should therefore lead to impairments in the single mutant. Ad 2 The dramatic phenotype of the tpt/xpt double mutant was the focus of the project and had to be analysed using the following approaches: Studies of metabolic fluxes using 15N and 13C labelling, analysis of subcellular distribution of metabolites after non-aqueous fractionation, detailed determination of photosynthetic parameters, generation of transgenic lines with an inducible repression of XPT in the background of the tpt mutant. The latter approach should be used to follow the phenotype development over time after induction of the repressor and combined with metabolome and transcriptome analyses during the kinetics after induction. Ad 3 The XPT is able to transport PEP in addition to the PPT. Since substantial PEP transport activities were detected in extracts of triple mutants despite knock-outs of both PPT genes and XPT, the role of both GPTs in PEP transport had to be investigated. Unfortunately, the objectives of the project were largely not accomplished.
