Inhibition of Arabidopsis growth by the allelopathic compound azetidine‐2‐carboxylate is due to the low amino acid specificity of cytosolic prolyl‐tRNA synthetase

Abstract

The toxicity of azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline, results from its incorporation into proteins due to misrecognition by prolyl-tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS-Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS-Cyt (At3g62120) is cytosolic. AtProRS-Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His-tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP-PPi exchange and aminoacylation assays, and showed similar K for L-proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS-Cyt showed nearly identical substrate specificity for L-proline and A2C, but for AtProRS-Org the specificity constant was 77.6 times higher for L-proline than A2C, suggesting that A2C-sensitivity may result from lower amino acid specificity of AtProRS-Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies.

Expression of genes for selected plant aminoacyl-tRNA synthetases in the abiotic stress

“…Research on plant aaRSs revealed their cellular localization (Rokov-Plavec et al 2008, Duchêne et al 2009, Kekez et al 2016, substrate specificity (Rokov et al 2008, Rokov and Weygand-Durasevic 2009, Rokov-Plavec et al 2002, 2004, Aldinger et al 2012, fidelity (Rokov-Plavec et al 2013, Lee et al 2016, Hoffman et al 2019 and protein interactors (Yang et al 2018, Kekez et al 2019. Thus far, only one crystal structure of plant aaRS is reported (Kekez et al 2019).…”

In vitro bioassay of allelopathy of <i>Arabidopsis thaliana</i> by sandwich method and protoplast co-culture method with digital image analysis

“…Allelopathic effects of co-cultivation with clover (Pedersen et al 2013), allelochemicals of rice (Kato-Noguchi and Kitajima 2015), and an analog of amino acid (Lee et al 2016), on the growth of A. thaliana have been reported. However, there have been few studies on the allelopathic activity of A. thaliana itself on other plants, except for the slight stimulatory effects of the seed exudates on cockscomb (Yokotani-Tomita et al 1998).…”

Quality control bytrans-editing factor prevents global mistranslation of non-protein amino acid α-aminobutyrate

“…43 Studies with Arabidopsis thaliana, a non-Aze-producing plant, indicate that differences in the properties and expression levels of cytoplasmic and organellular ProRSs may render a plant more susceptible to Aze misincorporation. 45 In A. thaliana, the cytoplasmic ProRS mischarges Aze more frequently than the organellular ProRS, making its roots, which produce more cytoplasmic ProRS than its shoots, more vulnerable to Aze toxicity. 45 These differences in ProRS quality control mechanisms thus make Aze an effective means of defense against competing plant species and predators.…”

“…45 In A. thaliana, the cytoplasmic ProRS mischarges Aze more frequently than the organellular ProRS, making its roots, which produce more cytoplasmic ProRS than its shoots, more vulnerable to Aze toxicity. 45 These differences in ProRS quality control mechanisms thus make Aze an effective means of defense against competing plant species and predators. 43,45 Prior to the present study, the only family of single domain trans-editing proteins known to target tRNAs misacylated with NPAs was the D-aminoacyl-tRNA deacylases (DTDs), which deacylate D-aa-tRNAs that form due to the inability of some ARSs to preferentially select the L-enantiomer.…”

“…45 These differences in ProRS quality control mechanisms thus make Aze an effective means of defense against competing plant species and predators. 43,45 Prior to the present study, the only family of single domain trans-editing proteins known to target tRNAs misacylated with NPAs was the D-aminoacyl-tRNA deacylases (DTDs), which deacylate D-aa-tRNAs that form due to the inability of some ARSs to preferentially select the L-enantiomer. 46 These enzymes are particularly important in bacteria that use D-Ala and D-Glu for peptidoglycan synthesis or for non-ribosomal synthesis of D-amino acid-containing antimicrobial peptides.…”