Characterization of Organelle Metabolite Transporters involved in Nucleotide Metabolism

• Applicant: Professor Dr. Claus-Peter Witte
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 452173586

Reactions of nucleotide metabolism take place in all organelles of the cell, and metabolic pathways of nucleotide metabolism can be distributed to different organelles. Some transporters that translocate metabolites of nucleotide metabolism across intracellular membranes have been described, but many transport processes are still not understood in molecular detail.This project aims to identify and characterize the plastid transporter for inosine monophosphate (IMP) - an end product of purine biosynthesis - and the plastid transporter for ribose - a product of nucleotide degradation - in both Phaseolus vulgaris and Arabidopsis thaliana. To identify possible transporters with these functions, a comparative analysis of the nodule and root transcriptomes of four legume species was carried out. Two legumes that export fixed nitrogen in the form of amides from the nodules and two legumes that export fixed nitrogen in the form of ureides were compared. Genes whose products are required in purine and ureide biosynthesis are likely to be active specifically in nodules from ureide producers. This assumption was confirmed on the basis of many known genes that are involved in these processes. Genes for possible IMP transporters (IMPEX) and a possible ribose transporter (RITAP) of P. vulgaris were found in this data set. Homologous genes were also identified in A. thaliana.Further investigations were initially carried out with genetic variants of Arabidopsis and showed that in an IMPEX mutant the concentrations of nucleotides are deregulated, which particularly affects IMP. Two independent RITAP mutants in Arabidopsis accumulated ribose, which was enhanced specifically in the mutants by external administration of uridine – a nucleoside hydrolyzed to uracil and ribose in the cytosol.In this project, these transporters are to be further characterized by metabolite analyses of genetic variants, also employing inducible lines in Arabidopsis. We will also investigate the function of the P. vulgaris transporters, including their role in ureide production, in transgenic adventitious roots (hairy roots) and nodules in which the corresponding genes were mutated by CRISPR / Cas9. In promoter-reporter and promoter-gene-reporter studies with fluorescent reporters in transgenic nodules, the tissue-specific promoter activity and the subcellular localization of the new transporters as well as of the known ureide biosynthetic enzymes will be investigated in order to develop a new model for the metabolic pathway of ureide biosynthesis in the nodule of P. vulgaris. Complementation studies with the plant transporters in mutants of Escherichia coli and studies on their substrate specificity and transport kinetics after expression in E. coli will complete the functional description.

• DFG Programme: Research Grants