Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

A plastid nucleoside kinase is involved in inosine salvage and control of purine nucleotide biosynthesis.

In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and cytidine have been characterized from many organisms, but kinases for inosine and guanosine salvage are not yet known in eukaryotes and only a few such enzymes have been described from bacteria. Here we identified Arabidopsis thaliana PLASTID NUCLEOSIDE KINASE 1 (PNK1), an enzyme highly conserved in plants and green algae belonging to the Phosphofructokinase B family. We demonstrate that PNK1 from A. thaliana is located in plastids and catalyzes the phosphorylation of inosine, 5-aminoimidazole-4-carboxamide-1-ß-d-ribose (AICA ribonucleoside), and uridine but not guanosine in vitro, and is involved in inosine salvage in vivo. PNK1 mutation leads to increased flux into purine nucleotide catabolism and, especially in the context of defective uridine degradation, to over-accumulation of uridine and UTP as well as growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. We additionally report that cold stress leads to accumulation of purine nucleotides, probably by inducing nucleotide biosynthesis, but that this adjustment of nucleotide homeostasis to environmental conditions is not controlled by PNK1.

N4-acetylation of cytidine in mRNA plays essential roles in plants.

The biological function of RNA can be modulated by base modifications. Here, we unveiled the occurrence of N4-acetylation of cytidine in plant RNA, including mRNA, by employing LC-MS/MS and acRIP-seq. We identified 325 acetylated transcripts from the leaves of 4-week-old Arabidopsis (Arabidopsis thaliana) plants and determined that 2 partially redundant N-ACETYLTRANSFERASEs FOR CYTIDINE IN RNA (ACYR1 and ACYR2), which are homologous to mammalian NAT10, are required for acetylating RNA in vivo. A double-null mutant was embryo lethal, while eliminating 3 of the 4 ACYR alleles led to defects in leaf development. These phenotypes could be traced back to the reduced acetylation and concomitant destabilization of the transcript of TOUGH, which is required for miRNA processing. These findings indicate that N4-acetylation of cytidine is a modulator of RNA function with a critical role in plant development and likely many other processes.

Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6f complex.

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

The nucleotide metabolome of germinating Arabidopsis thaliana seeds reveals a central role for thymidine phosphorylation in chloroplast development.

Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance.

Pyrimidine catabolism is required to prevent the accumulation of 5-methyluridine in RNA.

5-Methylated cytosine is a frequent modification in eukaryotic RNA and DNA influencing mRNA stability and gene expression. Here we show that free 5-methylcytidine (5mC) and 5-methyl-2'-deoxycytidine are generated from nucleic acid turnover in Arabidopsis thaliana, and elucidate how these cytidines are degraded, which is unclear in eukaryotes. First CYTIDINE DEAMINASE produces 5-methyluridine (5mU) and thymidine which are subsequently hydrolyzed by NUCLEOSIDE HYDROLASE 1 (NSH1) to thymine and ribose or deoxyribose. Interestingly, far more thymine is generated from RNA than from DNA turnover, and most 5mU is directly released from RNA without a 5mC intermediate, since 5-methylated uridine (m5U) is an abundant RNA modification (m5U/U ∼1%) in Arabidopsis. We show that m5U is introduced mainly by tRNA-SPECIFIC METHYLTRANSFERASE 2A and 2B. Genetic disruption of 5mU degradation in the NSH1 mutant causes m5U to occur in mRNA and results in reduced seedling growth, which is aggravated by external 5mU supplementation, also leading to more m5U in all RNA species. Given the similarities between pyrimidine catabolism in plants, mammals and other eukaryotes, we hypothesize that the removal of 5mU is an important function of pyrimidine degradation in many organisms, which in plants serves to protect RNA from stochastic m5U modification.

The novel chloroplast glucose transporter pGlcT2 affects adaptation to extended light periods.

Intracellular sugar compartmentation is critical in plant development and acclimation to challenging environmental conditions. Sugar transport proteins are present in plasma membranes and in membranes of organelles such as vacuoles, the Golgi apparatus, and plastids. However, there may exist other transport proteins with uncharacterized roles in sugar compartmentation. Here we report one such novel transporter of the Monosaccharide Transporter Family, the closest phylogenetic homolog of which is the chloroplast-localized glucose transporter pGlcT and that we therefore term plastidic glucose transporter 2 (pGlcT2). We show, using gene-complemented glucose uptake deficiency of an Escherichia coli ptsG/manXYZ mutant strain and biochemical characterization, that this protein specifically facilitates glucose transport, whereas other sugars do not serve as substrates. In addition, we demonstrate pGlcT2-GFP localized to the chloroplast envelope and that pGlcT2 is mainly produced in seedlings and in the rosette center of mature Arabidopsis plants. Therefore, in conjunction with molecular and metabolic data, we propose pGlcT2 acts as a glucose importer that can limit cytosolic glucose availability in developing pGlcT2-overexpressing seedlings. Finally, we show both overexpression and deletion of pGlcT2 resulted in impaired growth efficiency under long day and continuous light conditions, suggesting pGlcT2 contributes to a release of glucose derived from starch mobilization late in the light phase. Together, these data indicate the facilitator pGlcT2 changes the direction in which it transports glucose during plant development and suggest the activity of pGlcT2 must be controlled spatially and temporarily in order to prevent developmental defects during adaptation to periods of extended light.

Nucleotides and nucleotide derivatives as signal molecules in plants.

In reaction to a stimulus, signaling molecules are made, generate a response, and are then degraded. Nucleotides are classically associated with central metabolism and nucleic acid biosynthesis, but there are a number of nucleotides and nucleotide derivatives in plants to which this simple definition of a signaling molecule applies in whole or at least in part. These include cytokinins, chloroplast guanosine tetraposphate (ppGpp), as well as extracellular canonical nucleotides such as extracellular adenosine triphosphate (eATP) or nicotinamide adenine dinucleotide (eNAD+). In addition, there is a whole series of compounds derived from NAD+ such as adenosine diphosphate (ADP) ribose (ADPR), ATP-ADPR dinucleotides and their hydrolysis products (e.g. pRib-AMP) as well as different variants of cyclic ADPR (cADPR, 2'-cADPR, 3'-cADPR). Furthermore, cyclic nucleotides such as 3',5'-cAMP or 2',3'-cyclic nucleoside monophosphates. Interestingly, some of these compounds have recently been shown to play a central role in pathogen defense. In this review, we highlight these exciting new developments. We also review nucleotide derivatives that are considered candidates for signaling molecules, for example purine deoxynucleosides, for which the evidence base is still rather thin, and discuss more controversial cases.

Enhanced nucleotide analysis enables the quantification of deoxynucleotides in plants and algae revealing connections between nucleoside and deoxynucleoside metabolism.

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purine nucleotides.

In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.

A Kinase and a Glycosylase Catabolize Pseudouridine in the Peroxisome to Prevent Toxic Pseudouridine Monophosphate Accumulation.

Pseudouridine (Ψ) is a frequent nucleoside modification that occurs in both noncoding RNAs and mRNAs. In pseudouridine, C5 of uracil is attached to the Rib via an unusual C-glycosidic bond. This RNA modification is introduced on the RNA by site-specific transglycosylation of uridine (U), a process mediated by pseudouridine synthases. RNA is subject to constant turnover, releasing free pseudouridine, but the metabolic fate of pseudouridine in eukaryotes is unclear. Here, we show that in Arabidopsis (Arabidopsis thaliana), pseudouridine is catabolized in the peroxisome by (1) a pseudouridine kinase (PUKI) from the PfkB family that generates 5'-pseudouridine monophosphate (5'-ΨMP) and (2) a ΨMP glycosylase (PUMY) that hydrolyzes ΨMP to uracil and ribose-5-phosphate. Compromising pseudouridine catabolism leads to strong pseudouridine accumulation and increased ΨMP content. ΨMP is toxic, causing delayed germination and growth inhibition, but compromising pseudouridine catabolism does not affect the Ψ/U ratios in RNA. The bipartite peroxisomal PUKI and PUMY are conserved in plants and algae, whereas some fungi and most animals (except mammals) possess a PUMY-PUKI fusion protein, likely in mitochondria. We propose that vacuolar turnover of ribosomal RNA produces most of the pseudouridine pool via 3'-ΨMP, which is imported through the cytosol into the peroxisomes for degradation by PUKI and PUMY, a process involving a toxic 5'-ΨMP intermediate.

Calcium-Dependent Protein Kinase CPK1 Controls Cell Death by In Vivo Phosphorylation of Senescence Master Regulator ORE1.

Calcium-regulated protein kinases are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis (Arabidopsis thaliana) ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet-undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1 In addition, the decision to die is superimposed by an additional layer of control toward ORE1 via its posttranslational modification linked to the calcium-regulatory network through CPK1.

Structural basis for the substrate specificity and catalytic features of pseudouridine kinase from Arabidopsis thaliana.

RNA modifications can regulate the stability of RNAs, mRNA-protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/ß protein with a protruding small ß-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small ß-strand domain of the adjacent monomer. Conformational transition of the dimerized small ß-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis.

AMP and GMP Catabolism in Arabidopsis Converge on Xanthosine, Which Is Degraded by a Nucleoside Hydrolase Heterocomplex.

Plants can fully catabolize purine nucleotides. A firmly established central intermediate is the purine base xanthine. In the current widely accepted model of plant purine nucleotide catabolism, xanthine can be generated in various ways involving either inosine and hypoxanthine or guanosine and xanthosine as intermediates. In a comprehensive mutant analysis involving single and multiple mutants of urate oxidase, xanthine dehydrogenase, nucleoside hydrolases, guanosine deaminase, and hypoxanthine guanine phosphoribosyltransferase, we demonstrate that purine nucleotide catabolism in Arabidopsis (Arabidopsis thaliana) mainly generates xanthosine, but not inosine and hypoxanthine, and that xanthosine is derived from guanosine deamination and a second source, likely xanthosine monophosphate dephosphorylation. Nucleoside hydrolase 1 (NSH1) is known to be essential for xanthosine hydrolysis, but the in vivo function of a second cytosolic nucleoside hydrolase, NSH2, is unclear. We demonstrate that NSH1 activates NSH2 in vitro and in vivo, forming a complex with almost two orders of magnitude higher catalytic efficiency for xanthosine hydrolysis than observed for NSH1 alone. Remarkably, an inactive NSH1 point mutant can activate NSH2 in vivo, fully preventing purine nucleoside accumulation in nsh1 background. Our data lead to an altered model of purine nucleotide catabolism that includes an NSH heterocomplex as a central component.

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

m6A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N6-Methyl-AMP Deaminase in Plant and Mammalian Cells.

N6-methylated adenine (m6A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N6-methylated AMP (N6-mAMP), which has an unclear metabolic fate. We show that Arabidopsis thaliana and human cells require an N6-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N6-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N6A-RNA methylation in these organisms. MAPDA likely protects RNA from m6A misincorporation. This is required because eukaryotic RNA polymerase can use N6-mATP as a substrate. Upon abrogation of MAPDA, root growth is slightly reduced, and the N6-methyladenosine, N6-mAMP, and N6-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m6A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N6-mAMP was severalfold more abundant than N6-mATP in MAPDA mutants, we speculate that additional molecular filters suppress the generation of N6-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N6-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N6-mAMP accumulation and salvage.

Crystal structure and mutational analyses of ribokinase from Arabidopsis thaliana.

Nitrogen remobilization is a key issue in plants. Recent studies in Arabidopsis thaliana have revealed that nucleoside catabolism supplies xanthine, a nitrogen-rich compound, to the purine ring catabolic pathway, which liberates ammonia from xanthine for reassimilation into amino acids. Similarly, pyrimidine nuclosides are degraded and the pyrimidine bases are fully catabolized. During nucleoside hydrolysis, ribose is released, and ATP-dependent ribokinase (RBSK) phosphorylates ribose to ribose-5'-phosphate to allow its entry into central metabolism recycling the sugar carbons from nucleosides. In this study, we report the crystal structure of RBSK from Arapidopsis thaliana (AtRBSK) in three different ligation states: an unliganded state, a ternary complex with ribose and ATP, and a binary complex with ATP in the presence of Mg2+. In the monomeric conformation, AtRBSK is highly homologous to bacterial RBSKs, including the binding sites for a monovalent cation, ribose, and ATP. Its dimeric conformation, however, does not exhibit the noticeable ligand-induced changes that were observed in bacterial orthologs. Only in the presence of Mg2+, ATP in the binary complex adopts a catalytically competent conformation, providing a mode of action for Mg2+ in AtRBSK activity. The structural data combined with activity analyses of mutants allowed assignment of functional roles for the active site residues. Overall, this study provides the first structural characterization of plant RBSK, and experimentally validates a previous hypothetical model concerning the general reaction mechanism of RBSK.

The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.

The ribokinases of Arabidopsis thaliana and Saccharomyces cerevisiae are required for ribose recycling from nucleotide catabolism, which in plants is not essential to survive prolonged dark stress.

Nucleotide catabolism in Arabidopsis thaliana and Saccharomyces cerevisiae leads to the release of ribose, which requires phosphorylation to ribose-5-phosphate mediated by ribokinase (RBSK). We aimed to characterize RBSK in plants and yeast, to quantify the contribution of plant nucleotide catabolism to the ribose pool, and to investigate whether ribose carbon contributes to dark stress survival of plants. We performed a phylogenetic analysis and determined the kinetic constants of plant-expressed Arabidopsis and yeast RBSKs. Using mass spectrometry, several metabolites were quantified in AtRBSK mutants and double mutants with genes of nucleoside catabolism. Additionally, the dark stress performance of several nucleotide metabolism mutants and rbsk was compared. The plant PfkB family of sugar kinases forms nine major clades likely representing distinct biochemical functions, one of them RBSK. Nucleotide catabolism is the dominant ribose source in plant metabolism and is highly induced by dark stress. However, rbsk cannot be discerned from the wild type in dark stress. Interestingly, the accumulation of guanosine in a guanosine deaminase mutant strongly enhances dark stress symptoms. Although nucleotide catabolism contributes to carbon mobilization upon darkness and is the dominant source of ribose, the contribution appears to be of minor importance for dark stress survival.