Several key enzymatic steps in Nucleotide metabolism have been subject of studies over the last years in our group. These cover the pathway branches of salvage,(Hickl et al., 2021) catabolism and de novo synthesis. Currently, the enzymes Cytidine-Triphosphate Synthase (CTPS) and Inosine Monophosphate Dehydrogenase (IMPDH) are in the focus of our work. CTPS is the sole enzyme converting uridylates to cytidylates by catalyzing the reaction of UTP + ATP + Gln = CTP + ADP + Glu, activated by GTP. CTPS2 (out of 5 isoforms in Arabidopsis) was shown to be important for early embryo development and Nucleotide homeostasis especially in Chloroplasts. CTPS4 was adjudicated a function in drought resistance.

IMPDH controls the purine synthesis pathway branch to guanine nucleotides. IMPDH together with CTPS can be seen as important steps to balance nucleotide pools in the plant cytosol.

This work is funded by the Deutsche Forschungsgemeinschaft (Mo 1032/5-2)

Bellin L, Scherer V, Dörfer E, Lau A, Vicente AM, Meurer J, Hickl D, Möhlmann T (2021) Cytosolic CTP production limits the establishment of photosynthesis in Arabidopsis. Front Plant Sci 12: 789189 10.3389/fpls.2021.789189

Hickl D, Drews F, Girke C, Zimmer D, Mühlhaus T, Hauth J, Nordström K, Trentmann O, Neuhaus EH, Scheuring D, Fehlmann T, Keller A, Simon M, Möhlmann T (2021) Differential degradation of RNA species by autophagy-related pathways in Arabidopsis. J Exp Bot 72: 6867-6881 10.1093/jxb/erab321

Hickl D, Scheuring D, Möhlmann T (2021) CTP Synthase 2 From Arabidopsis thaliana Is Required for Complete Embryo Development. Front Plant Sci 12: 652434 10.3389/fpls.2021.652434

Krämer M, Dorfer E, Hickl D, Bellin L, Scherer V, Möhlmann T (2022) Cytidine Triphosphate Synthase Four From Arabidopsis thaliana Attenuates Drought Stress Effects. Front Plant Sci 13: 842156 10.3389/fpls.2022.842156

Levels of free Nucleotides are kept constant and are balanced between purines and pyrimidines. The consequences of nucleotide imbalance or limitation are intensively studied in mammalian systems, other eukaryotes and prokaryotes. Yet, studies of this pathway in plants are scarce, especially with respect to its regulation and interactions with other pathways. Ribosomal RNA pools for example, dynamically respond to growth signals and during acclimation to cold. This response in RNA synthesis is controlled by target of rapamycin (TOR) by inducing expression of de novo synthesis genes aspartate transcarbamoylase (ATC) and dihydroorotate dehydrogenase (DHODH).

ATC catalyzes the first committed step in pyrimidine de novo synthesis. By studying the structure function interaction of ATC from Arabidopsis, the molecular mechanism of feedback inhibition by UMP, uniquely found in plants, was clarified. When reducing ATC amount in corresponding amiRNA mutants, a nucleotide imbalance was observed , leading to impaired photosynthesis, growth and cold acclimation.

Uracil phosphoribosyltransferase (UPP) catalyzes the conversion of uracil to UMP in the chloroplast. Surprisingly, a moonlighting function of UPP in the establishment of photosynthesis was observed, explaining seedling lethality of corresponding knock-out mutants. Apparently, UPP is required for proper establishment of the cytochrome b6/f complex. Elucidation of the exact molecular mechanism behind this observation is subject of current studies in our group.

This work is funded by the Deutsche Forschungsgemeinschaft within the Transregional Research Initiative TRR175 (www.tr175.bio.lmu.de), and the BioComp 4.0 Research Initiative of the Country Rhineland Palatinate (https://rptu.de/biocomp/biocomp-40).

Bellin L, Del Cano-Ochoa F, Velazquez-Campoy A, Möhlmann T, Ramon-Maiques S (2021) Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase. Nat Commun 12: 947 10.1038/s41467-021-21165-9

Bellin L, Garza Amaya DL, Scherer V, Pruss T, John A, Richter A, Möhlmann T (2023) Nucleotide imbalance, provoked by downregulation of Aspartate Transcarbamoylase impairs cold acclimation in Arabidopsis. Molecules 2810.3390/molecules28041585

Bellin L, Melzer M, Hilo A, Garza Amaya DL, Keller I, Meurer J, Mohlmann T (2023) Nucleotide Limitation Results in Impaired Photosynthesis, Reduced Growth and Seed Yield Together with Massively Altered Gene Expression. Plant Cell Physiol 64: 1494-1510 10.1093/pcp/pcad063

Scherer V, Bellin L, Schwenkert S, Lehmann M, Rinne J, Witte CP, Jahnke K, Richter A, Pruss T, Lau A, Waller L, Stein S, Leister D, Möhlmann T (2024). UPP affects chloroplast development by interfering with chloroplast proteostasis. BioRxiv. doi: 10.1101/2023.08.28.555145

Schwenkert S, Fernie AR, Geigenberger P, Leister D, Möhlmann T, Naranjo B, Neuhaus HE (2022) Chloroplasts are key players to cope with light and temperature stress. Trends Plant Sci 27: 577-587 10.1016/j.tplants.2021.12.004

For a long time we studied transport proteins for nucleosides and nucleobases, belonging to the protein families of Equilibrative Nucleoside Transport (ENT), Nucleobase:Cation Symport 1 (NCS1), and Nucleobase Ascorbate Transport (NAT) belonging to Nucleobase:Cation Symport 2 (NCS2). The main ENT members out of 8 in Arabidopsis were ENT1 (vacuole) and ENT3 (plasma membrane). Besides distribution of nucleosides within the plant and uptake from the rhizosphere, a role in plant pathogen interaction and cytokinin transport were adjudicated to these proteins. PLUTO, the sole NCS1 protein in Arabidopsis exhibits nucleobase (uracil, adenine, guanine) transport activity and resides in the inner chloroplast envelope. From the NCS2/NAT protein we studied NAT3 and NAT12 in detail and found they catalyze high affinity, proton driven nucleobase transport and localize to the plasma membrane.

Girke C, Daumann M, Niopek-Witz S, Möhlmann T (2014) Nucleobase and nucleoside transport and integration into plant metabolism. Front Plant Sci 5: 443 10.3389/fpls.2014.00443 [doi]

Niopek-Witz S, Deppe J, Lemieux MJ, Möhlmann T (2014) Biochemical characterization and structure-function relationship of two plant NCS2 proteins, the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana. Biochim Biophys Acta 1838: 3025-3035 S0005-2736(14)00295-8 [pii];10.1016/j.bbamem.2014.08.013 [doi]

Witz S, Jung B, Fürst S, Möhlmann T (2012) De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis. Plant Cell 24: 1549-1559 tpc.112.096743

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