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.

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 DEHYDRO-GENASE 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.

Inosine is deaminated adenosine. Inosine is enzymatically introduced in some plant 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 non-enzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (ITP / dITP) during nucleic acid synthesis. We identified a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) which is conserved in many organisms. Arabidopsis ITPA dephosphorylates deaminated nucleoside di- and triphosphates to their respective monophosphates. ITPA mutation causes inosine di- and triphosphate accumulation in vivo and an elevated (deoxy)inosine content in DNA and RNA. Cadmium-induced oxidative stress, known to foster deamination, leads to more ITP in the wildtype and especially in itpa mutants suggesting that ITP originates from ATP deamination. By contrast, erroneous IMP phosphorylation by AMP kinases seems not to be a major ITP source in vivo although these enzymes phosphorylate IMP in vitro. Mutation of ITPA causes salicylic acid (SA) accumulation, early senescence and upregulation of transcripts associated with immunity and senescence. ITPA is part of a molecular protection system, preventing accumulation of (d)ITP, its usage for nucleic acid synthesis, and probably nucleic acid stress leading to SA accumulation, stress gene induction and early senescence.