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

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/β protein with a protruding small β-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 β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-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. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research

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

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

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 13C from labeling experiments with valine-13C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13C5, 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

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

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

Coprophagous features in carnivorous Nepenthes plants: A task for ureases

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy

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