Aldehydes generate N-alkylated-aa-tRNA adducts

TLC showing modification on L- and D-Tyr-tRNATyr by A) formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, isovaleraldehyde, decanal and B) MG (AMP: adenine mono phosphate which corresponds to free tRNA; whereas Tyr-AMP and Modified-Tyr-AMP corresponds to unmodified and modified Tyr-tRNATyr). Mass spectra showing C) D-Phe-tRNAPhe, D) formaldehyde modified D-Phe-tRNAPhe E) propionaldehyde modified D-Phe-tRNAPhe F) Butyraldehyde modified D-Phe-tRNAPhe G) MG modified D-Phe-tRNAPhe. H) Graph showing the effect of increasing chain length of aldehyde on modification propensity with aa-tRNA at two different concentrations of various aldehydes. Effect of I) formaldehyde, and J) MG modification on stability of ester linkage in D-aa-tRNA under alkaline conditions.

DTD2 acts as a general aldehyde detoxification system

Deacylation assays on formaldehyde, propionaldehyde, methylglyoxal and butyraldehyde modified D-Tyr-tRNATyr substrates by AtDTD2 (A-D), PhoDTD2 (E-H), AtDTD1 (I-L): M) Table showing the effective activity concentration of AtDTD2, PhoDTD2, AtDTD1, archaeal PTH and bacterial PTH that completely deacylates aldehyde modified D-Tyr-tRNATyr (‘-’ denotes no activity; *from Mazeed et al.40).

DTD2 mutant plants are susceptible to physiologically abundant toxic aldehydes

A) Schematics showing the site of T-DNA insertion in (SAIL_288_B09) the first exon of DTD2 gene and RT-PCR showing the expression of DTD2 gene in wildtype (Wt), dtd2-/-, dtd2 /-::AtDTD2 (rescue) and dtd2-/-::AtDTD2 H150A (catalytic mutant) plants lines used in the study. B) Toxicity assays showing the effect of formaldehyde and MG with and without D-amino acid (D-Tyrosine (D-Tyr)) on dtd2-/- plants. Graph showing effect of C) Murashige and Skoog agar (MSA), D) 1.5 mM MG, E) 0.5 mM D-Tyr and 1.5 mM MG, F) 0.5 mM formaldehyde and G) 0.5 mM D-Tyr and 0.5 mM formaldehyde on growth of Wt (Blue), dtd2-/-(Green), dtd2-/-::AtDTD2 H150A (catalytic mutant) (Purple), and dtd2-/-::AtDTD2 (rescue) (Red) plants. Cotyledon surface area (mm2) is plotted as parameter for seedling size (n=4-15). P values less than 0.05 are denoted as ns and P ≤ 0.001 are denoted as ***. H) Graph showing effect of formaldehyde, and MG on germination of Wt, dtd2-/-, dtd2-/-::AtDTD2 (rescue) and dtd2-/-::AtDTD2 H150A (catalytic mutant) plants.

Overexpression of DTD2 confers increased multi aldehyde tolerance to Arabidopsis thaliana

DTD2 overexpression (OE) plants grow better than wild type Col-0 under A) 0.5 mM, 0.75 mM, 1.0 mM and 1.25 mM of formaldehyde with and without 0.5 mM D-tyrosine. Cotyledon surface area (mm2) is plotted as parameter for seedling size (n=5-15); P values less than 0.05 are denoted as ns and P ≤ 0.001 are denoted as ***. B) Growth of DTD2 OE and wild type Col-0 under 0.5 mM, 0.75 mM, 1.0 mM, 1.25 mM, 1.5 mM of MG and 0.5 mM, 0.75 mM, 1.0 mM MG with 0.5 mM D-tyrosine. C) The qPCR analysis showing fold change of DTD2 gene expression in DTD2 OE plant line used.

Terrestrialization of plants is associated with expansion of aldehyde metabolising genes

Deacylation assays of KnDTD2 on A) formaldehyde, B) propionaldehyde and C) MG modified D-Tyr-tRNATyr. D) Table showing the presence of 31 genes associated with formaldehyde metabolism in all KEGG organisms across life forms. E) Model showing the expansion of aldehyde metabolising repertoire, cell wall components and recruitment of archaeal DTD2 in charophytes during land plant evolution.

DTD2 acts as a general aldehyde detoxifier in land plants during translation quality control

Model showing the production of multiple aldehydes like formaldehyde, acetaldehyde and methylglyoxal (MG) through various metabolic processes in plants which generate stable alkyl modification on D-aminoacyl-tRNA adducts and DTD2 is unique proofreader for these alkyl adducts thereby protecting the plants from aldehyde toxicity associated with translation apparatus emerged from expanded metabolic pathways and D-amino acids.

Aldehydes modify the amino group of amino acids in D-aa-tRNAs

A) Table showing the presence of various aldehydes in different domains of life3,21–28. B) Table showing modification type, expected and observed mass change by different aldehyde on D-aa-tRNA; *Mass change observed after acetaldehyde treatment in earlier study40. Tandem fragmentation mass spectra (MS2) showing modification on the amino group of amino acid in C) D-Phe-tRNAPhe, D) formaldehyde modified D-Phe-tRNAPhe, E) propionaldehyde modified D-Phe-tRNAPhe, F) butyraldehyde modified D-Phe-tRNAPhe and G) MG modified D-Phe-tRNAPhe. H) Table showing the calculated molecular size and modification size (both volume Å3 and surface area Å2) by various aldehydes on D-amino acid (D-alanine) using the Voss Volume Voxelator (3V) webserver at probe 1.5 Å radius. I) Thin layer chromatography showing the activation profile of EF-Tu via RNase protection-based assay. J) Thin layer chromatography showing the formaldehyde modification on L- and D-aa-tRNAs in the presence of EF-Tu.

DTD2 is inactive on aldehyde-modified D-aa-tRNAs beyond three-carbon aldehyde chain length

Deacylation assays of AtDTD2 on A) valeraldehyde, B) isovaleraldehyde modified D-Tyr-tRNATyr; Deacylation assays of PhoDTD2 on C) valeraldehyde, D) isovaleraldehyde modified D-Tyr-tRNATyr; Deacylation assays of AtDTD1 on E) valeraldehyde, F) isovaleraldehyde modified D-Tyr-tRNATyr.

DTD2 acts as a general aldehyde detoxification system

Deacylation assays of AtDTD2 on A) formaldehyde, B) propionaldehyde, C) MG, D) butyraldehyde, E) valeraldehyde, and F) isovaleraldehyde modified L-Tyr-tRNATyr; Deacylation assays of PhoDTD2 on G) formaldehyde, H) propionaldehyde, I) MG, J) butyraldehyde, K) valeraldehyde, and L) isovaleraldehyde modified L-Tyr-tRNATyr; Deacylation assays of EcPTH on M) formaldehyde, and N) MG modified L-Tyr-tRNATyr; Deacylation assays of StPTH on O) formaldehyde, and P) MG modified L-Tyr-tRNATyr.

MG and formaldehyde inhibit the germination of DTD2 mutant plants

A) Toxicity assays showing the effect of 0.1% ethanol on wildtype (Wt), dtd2-/-, dtd2-/-::AtDTD2 H150A (catalytic mutant) and dtd2-/-::AtDTD2 (rescue) plants B) Graph showing cumulative effect of MG and D-Tyrosine on germination of Col-0, dtd2 (-/-), and dtd2 (-/-)::AtDTD2 (rescue) plants. C) Structure based sequence alignment showing invariant catalytic histidine in evolutionarily distinct DTD2 sequences. Deacylation of D) acetaldehyde modified D-Tyr-tRNATyr with PhoDTD2 and AtDTD2. E) PhoDTD2 H140A and AtDTD2 H150A (catalytic mutants) and F) Deacylation of acetaldehyde modified L-Tyr-tRNATyr with PhoDTD2 H140A and AtDTD2 H150A (catalytic mutants).

Overexpression of DTD2 confers multi aldehyde tolerance with D-amino acid stress in Arabidopsis thaliana.

DTD2 overexpression line grows better than wild type Col-0 under A) no stress, B) 0.75 mM formaldehyde and C) 1.25 mM MG. D) Graph showing the seedling size of Wt and DTD2 OE plants under 0.5 mM, 1.25 mM formaldehyde and 0.5 mM D-tyrosine with 0.5 mM formaldehyde. Cotyledon surface area (mm2) is plotted as parameter for seedling size (n=4-15); P values less than 0.05 are denoted as ns and P ≤ 0.001 are denoted as ***.

Land plant evolution is associated with the expansion of aldehyde metabolising repertoire

Deacylation assays of KnDTD2 on A) butyraldehyde, B) valeraldehyde, and C) isovaleraldehyde modified D-Tyr-tRNATyr. D) Graph showing the number of genes for aldehyde oxidase (AOX) in viridiplantae. E) Schematics showing multiple enzymes involved in formaldehyde metabolism based on KEGG database across life forms. F) Table showing the presence of enzymes involved in MG production in KEGG genomes of opisthokonts, land plants, algae, bacteria and archaea. Number denotes the percentage of KEGG organism encoding respective enzymes. G) Table showing the emergence of enzymes responsible for pectin biosynthesis and degradation in land plant ancestors. (*Present in few chlorophytes).

List of primers used in the study