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.

Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement.

The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

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.

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.

Enzymes and cellular interplay required for flux of fixed nitrogen to ureides in bean nodules.

Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.

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.

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP).

This protocol describes the isolation of mitochondria by affinity chromatography using magnetic beads coated with Strep-Tactin in a timeframe of ca. 30 min. Compared to a classic differential and density gradient centrifugation this protocol enables a more rapid and efficient isolation of mitochondria even with small amounts of plant material. Transgenic plants with mitochondria that are decorated with a protein that is integrated into the outer mitochondrial membrane and fused to a green fluorescent protein (GFP) and a TwinStrep-tag facing the cytosol. This tag can bind to Strep-Tactin coated magnetic beads. Isolated mitochondria still bound to magnetic beads are uniquely suited for measuring oxygen consumption rates since this measurement needs mitochondria to be immobilized on the bottom of the measuring well. Furthermore, the isolated mitochondria can be used for downstream applications such as proteomics and metabolomics. This technique also allows for the isolation of mitochondria from specific cell types and tissues by altering the expression of the protein decorating the mitochondria.

Purification and Analysis of Nucleotides and Nucleosides from Plants.

This protocol describes necessary steps to isolate and quantify nucleotides and nucleosides from plant samples. Proper sample preparation in combination with liquid chromatography coupled to mass spectrometry enables the sensitive detection and quantification of metabolites of low abundance. Utilizing a liquid-liquid extraction in combination with a weak anion-exchange solid phase extraction enables the separation of negatively charged molecules from uncharged metabolites or cations.

Calmodulin-like protein CML24 interacts with CAMTA2 and WRKY46 to regulate ALMT1-dependent Al resistance in Arabidopsis thaliana.

ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1)-mediated malate exudation from roots is critical for aluminium (Al) resistance in Arabidopsis. Its upstream molecular signalling regulation is not yet well understood. The role of CALMODULIN-LIKE24 (CML24) in Al-inhibited root growth and downstream molecular regulation of ALMT1-meditaed Al resistance was investigated. CML24 confers Al resistance demonstrated by an increased root-growth inhibition of the cml24 loss-of-function mutant under Al stress. This occurs mainly through the regulation of the ALMT1-mediated malate exudation from roots. The mutation and overexpression of CML24 leads to an elevated and reduced Al accumulation in the cell wall of roots, respectively. Al stress induced both transcript and protein abundance of CML24 in root tips, especially in the transition zone. CML24 interacts with CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2 (CAMTA2) and promotes its transcriptional activity in the regulation of ALMT1 expression. This results in an enhanced malate exudation from roots and less root-growth inhibition under Al stress. Both CML24 and CAMTA2 interacted with WRKY46 suppressing the transcriptional repression of ALMT1 by WRKY46. The study provides novel insights into understanding of the upstream molecular signalling of the ALMT1-depdendent Al resistance.

Initiation of cytosolic plant purine nucleotide catabolism involves a monospecific xanthosine monophosphate phosphatase.

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo.

Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants.

In this study, we describe the establishment of the knockout marker gene MAR1 for selection of CRISPR/Cas9-edited Arabidopsis seedlings and tomato explants in tissue culture. MAR1 encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also opportunistically transports aminoglycoside antibiotics into these organelles and defects of the gene render plants insensitive to those compounds. Here, we show that mutations of MAR1 induced by the CRISPR system confer kanamycin-resistance to Arabidopsis plants and tomato tissues. MAR1 is single-copy in a variety of plant species and the corresponding proteins form a distinct phylogenetic clade allowing easy identification of MAR1 orthologs in different plants. We demonstrate that in multiplexing approaches, where Arabidopsis seedlings were selected via a CRISPR/Cas9-induced kanamycin resistance mediated by MAR1 mutation, a mutation in a second target gene was observed with higher frequency than in a control population only selected for the presence of the transgene. This so called co-selection has not been shown before to occur in plants. The technique can be employed to select for edited plants, which might be particularly useful if editing events are rare.

Analysis of Nucleosides and Nucleotides in Plants: An Update on Sample Preparation and LC-MS Techniques.

Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid-liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites.

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.

Improving plant drought tolerance and growth under water limitation through combinatorial engineering of signalling networks.

Agriculture is by far the biggest water consumer on our planet, accounting for 70 per cent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently ('more crop per drop'). Water-use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here, we describe a combinatorial engineering approach to optimize signalling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium-dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water-limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water-limited conditions. Our work provides an efficient strategy for engineering complex signalling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function.

Author Correction: RNA-Seq reveals novel genes and pathways associated with hypoxia duration and tolerance in tomato root.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of nitric oxide (NO)-responsive genes under hypoxia in tomato (Solanum lycopersicum L.) root.

Flooding periods, as one probable consequence of climate change, will lead more frequently to plant hypoxic stress. Hypoxia sensing and signaling in the root, as the first organ encountering low oxygen, is therefore crucial for plant survival under flooding. Nitric oxide has been shown to be one of the main players involved in hypoxia signaling through the regulation of ERFVII transcription factors stability. Using SNP as NO donor, we investigated the NO-responsive genes, which showed a significant response to hypoxia. We identified 395 genes being differentially regulated under both hypoxia and SNP-treatment. Among them, 251 genes showed up- or down-regulation under both conditions which were used for further biological analysis. Functional classification of these genes showed that they belong to different biological categories such as primary carbon and nitrogen metabolism (e.g. glycolysis, fermentation, protein and amino acid metabolism), nutrient and metabolites transport, redox homeostasis, hormone metabolism, regulation of transcription as well as response to biotic and abiotic stresses. Our data shed light on the NO-mediated gene expression modulation under hypoxia and provides potential targets playing a role in hypoxia tolerance. These genes are interesting candidates for further investigating their role in hypoxia signaling and survival.

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.

RNA-Seq reveals novel genes and pathways associated with hypoxia duration and tolerance in tomato root.

Due to climate change, economically important crop plants will encounter flooding periods causing hypoxic stress more frequently. This may lead to reduced yields and endanger food security. As roots are the first organ to be affected by hypoxia, the ability to sense and respond to hypoxic stress is crucial. At the molecular level, therefore, fine-tuning the regulation of gene expression in the root is essential for hypoxia tolerance. Using an RNA-Seq approach, we investigated transcriptome modulation in tomato roots of the cultivar 'Moneymaker', in response to short- (6 h) and long-term (48 h) hypoxia. Hypoxia duration appeared to have a significant impact on gene expression such that the roots of five weeks old tomato plants showed a distinct time-dependent transcriptome response. We observed expression changes in 267 and 1421 genes under short- and long-term hypoxia, respectively. Among these, 243 genes experienced changed expression at both time points. We identified tomato genes with a potential role in aerenchyma formation which facilitates oxygen transport and may act as an escape mechanism enabling hypoxia tolerance. Moreover, we identified differentially regulated genes related to carbon and amino acid metabolism and redox homeostasis. Of particular interest were the differentially regulated transcription factors, which act as master regulators of downstream target genes involved in responses to short and/or long-term hypoxia. Our data suggest a temporal metabolic and anatomic adjustment to hypoxia in tomato root which requires further investigation. We propose that the regulated genes identified in this study are good candidates for further studies regarding hypoxia tolerance in tomato or other crops.

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.