The identification and characterisation of a ribokinase and a putative D-ribose permease in Arabidopsis thaliana

Abstract

Plants are stationary organisms that rely on the e cient uptake and remobilization of nutrients for growth and reproduction. One of the most abundant nutrients is nitrogen (N), of which the majority is located in proteins, however. N can also be found in nucleotides as part of the purine and pyrimidine nucleobases and can furthermore be recycled by the nucleotide catabolism pathway. During the degradation of ribo-nucleosides, derived from RNA, the enzyme nucleoside hydrolase 1 (NSH1) hydrolyses the N-glycosidic bond between the nucleobase and the D-ribose residue. The N from the nucleobase is then recycled to ammonia in a multi-step process via uric acid and allantoin. The process of D-ribose recycling is unknown up to now. Plant lines of mutant genes involved in this purine nucleotide catabolism, like the guanosine deaminase (GSDA), show a necrotic phenotype under prolonged dark stress conditions, suggesting that carbon starvation, due to the lack of recycled D-ribose, could lead to this drastic phenotype. The enzyme responsible for the recycling of D-ribose is ribokinase (RBSK). It phosphorylates D-ribose to D-ribose 5-phosphate, which can be used afterwards in the non-oxidative pentose phosphate pathway, the nucleotide de novo synthesis or the nucleotide salvage reactions. In this study, the RBSK from Arabidopsis thaliana is described (AtRBSK) as the rst plant RBSK. The homologous enzyme from Saccharomyces cerevisiae was included into the analysis, because of contradicting results regarding the identi cation of yeast RBSK in a former study (Xu et al., 2013). The proteins were transiently produced in Nicotiana benthamiana with a C-terminal StrepII tag for protein puri cation and detection. For the evaluation of the kinetic constants, a HPLC kinase assay was developed and established. In the in vivo analysis, metabolites from double mutant lines of the purine and pyrimidine nucleotide metabolic pathways and the RBSK mutant line were extracted to clarify the contribution of nucleotide metabolism to the D-ribose pool in plants. A comprehensive dark stress experiment coupled with metabolite analysis by mass spectrometry, showed an impact of prolonged dark stress on nucleotide metabolism and the D-ribose pool in plants. Furthermore, it could be excluded that the lack of D-ribose is causing the gsda dark stress phenotype. In the second part of this work, candidate genes for a plastidic D-ribose transporter were found by comparative expression data analysis in legumes, linking cytosolic D-ribose, released by the nucleotide metabolism, with the plastidic D-ribose phosphorylation by RBSK. A promising candidate gene was found in pGLCT which is transcriptionally upregulated in a situation of high D-ribose turnover, as found in nodules of ureide exporting legumes. Furthermore, metabolite analysis in A. thaliana and transient overexpression in S. cerevisiae were used for the investigation of the role of pGLCT in D-ribose translocation. By investigating the plant RBSK from A. thaliana together with the homologous enzyme from S. cerevisiae and furthermore with pGLCT as the plastidic D-ribose transporter, the metabolic process of D-ribose recycling during purine nucleotide degradation was revealed.