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Kumar etal. eLife 2023;13:RP92827. DOI: https://doi.org/10.7554/eLife.92827 1 of 22
A translation proofreader of archaeal
origin imparts multi- aldehyde stress
tolerance to landplants
Pradeep Kumar1,2,3, Ankit Roy1, Shivapura Jagadeesha Mukul1,2,3,
Avinash Kumar Singh1, Dipesh Kumar Singh1, Aswan Nalli1, Pujaita Banerjee1,
Kandhalu Sagadevan Dinesh Babu1, Bakthisaran Raman1, Shobha P Kruparani1,
Imran Siddiqi1,2, Rajan Sankaranarayanan1,2,3*
1CSIR–Centre for Cellular and Molecular Biology, Hyderabad, India; 2Academy of
Scientific and Innovative Research (AcSIR), CSIR–CCMB Campus, Hyderabad, India;
3Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
Abstract Aldehydes, being an integral part of carbon metabolism, energy generation, and
signalling pathways, are ingrained in plant physiology. Land plants have developed intricate meta-
bolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh
terrestrial environments. Here, we show that physiologically produced aldehydes, i.e., formaldehyde
and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl- tRNAs, a substrate
for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems,
D- aminoacyl- tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively.
Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D- aminoacyl
adducts on tRNA thereby shielding archaea and plants from these system- generated aldehydes.
Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants
susceptible to these toxic aldehydes as they generate stable alkyl modification on D- aminoacyl-
tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde
metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of
archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection
against aldehydes than in wild type Arabidopsis highlighting its role as a multi- aldehyde detoxifier
that can be explored as a transgenic crop development strategy.
eLife assessment
The work is a fundamental contribution towards understanding the role of archaeal and plant
D- aminoacyl- tRNA deacylase 2 (DTD2) in deacylation and detoxification of D- Tyr- tRNATyr modified
by various aldehydes produced as metabolic byproducts in plants. It integrates convincing results
from both in vitro and in vivo experiments to address the long- standing puzzle of why plants outper-
form bacteria in handling reactive aldehydes and suggests a new strategy for stress- tolerant crops.
A limitation of the study is the lack of evidence for accumulation of toxic D- aminoacyl tRNAs and
impairment of translation in plant cells lacking DTD2.
Introduction
Reactive metabolites are an integral part of biological systems as they fuel a plethora of fundamental
processes of life. Metabolically generated aldehydes are chemically diverse reactive metabolites such
as formaldehyde (1- C), acetaldehyde (2- C), and methylglyoxal (MG; 3- C). Formaldehyde integrates
RESEARCH ARTICLE
*For correspondence:
sankar@ccmb.res.in
Competing interest: See page
17
Funding: See page 17
Sent for Review
26 September 2023
Preprint posted
10 October 2023
Reviewed preprint posted
24 November 2023
Reviewed preprint revised
05 February 2024
Version of Record published
19 February 2024
Reviewing Editor: Alan G
Hinnebusch, Eunice Kennedy
Shriver National Institute of Child
Health and Human Development,
United States
Copyright Kumar etal. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
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Figure 1. Aldehydes generate N- alkylated- aa- tRNA adducts. Thin- layer chromatography (TLC) showing modication on L- and D- Tyr- tRNATyr by (A)
formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, isovaleraldehyde, decanal and (B) MG (AMP: adenine monophosphate
which corresponds to free tRNA, whereas Tyr- AMP and modied- Tyr- AMP correspond to unmodied and modied Tyr- tRNATyr). These modications
were generated by incubating 2µM aa- tRNA with 100mM of respective aldehydes along with 20mM sodium cyanoborohydride (in 100mM potassium
acetate [pH 5.4]) as a reducing agent at 37°C for 30min. Mass spectra showing (C) D- Phe- tRNAPhe, (D)formaldehyde- modied D- Phe- tRNAPhe, (E)
propionaldehyde- modied D- Phe- tRNAPhe, (F) butyraldehyde- modied D- Phe- tRNAPhe, (G) MG- modied D- Phe- tRNAPhe. (H)Graph showing the effect
of increasing chain length of aldehyde on modication propensity with aa- tRNA at two different concentrations of various aldehydes (n=3). Effect of (I)
formaldehyde and (J) MG modication on stability of ester linkage in D- aminoacyl- tRNA (D- aa- tRNA) under alkaline conditions (n=3).
The online version of this article includes the following source data and gure supplement(s) for gure 1:
Figure 1 continued on next page
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various carbon metabolic pathways and is produced as a by- product of oxidative demethylation by
various enzymes (Jardine etal., 2017; Song etal., 2013; Trézl etal., 1998; Loenarz and Schofield,
2008; Shi et al., 2004; Walport et al., 2012) whereas acetaldehyde is an intermediate of anaer-
obic fermentation (Tadege and Kuhlemeier, 1997). Alternatively, MG is produced via the glycolysis
pathway from dihydroxyacetone phosphate and glyceraldehyde- 3- phosphate, oxidative deamina-
tion of glycine and threonine, fatty acid degradation, and auto- oxidation of glucose inside the cell
(Mostofa etal., 2018). These aldehydes are involved in carbon metabolism (Jardine etal., 2017;
Song etal., 2013; Trézl etal., 1998; Burgos- Barragan etal., 2017; Hill etal., 2011), energy gener-
ation (Tadege and Kuhlemeier, 1997), and signalling (Mostofa etal., 2018; Kosmachevskaya etal.,
2017), respectively, in all domains of life. In addition to the three aldehydes discussed above, plants
also produce a wide range of other aldehydes under various biotic and abiotic stresses (Mostofa
etal., 2018; Jardine etal., 2009). Despite their physiological importance, these aldehydes become
genotoxic and cellular hazards at higher concentrations as they irreversibly modify the free amino
group of various essential biological macromolecules like nucleic acids, proteins, lipids, and amino
acids (Seitz and Stickel, 2007; Fang and Vaca, 1997; Matsuda etal., 1999; Fang and Vaca, 1995;
Carlsson etal., 2014). Increased levels of formaldehyde and MG lead to toxicity in various life forms
like bacteria (Chen etal., 2016) and mammals (Burgos- Barragan et al., 2017; Pontel etal., 2015;
Allaman etal., 2015). However, archaea and plants possess these aldehydes in high amounts (>25-
fold) (Figure1—figure supplement 1A), yet there is no evidence of toxicity (Trézl etal., 1998; Miller
etal., 2017; Dingler etal., 2020; Li etal., 2017; Kimmerer and Macdonald, 1987; Quintanilla
etal., 2007; Yadav etal., 2005; Rabbani and Thornalley, 2014; Wang etal., 2019; Baskaran etal.,
1989). This suggests that both archaea and plants have evolved specialised protective mechanisms
against toxic aldehyde flux.
Using genetic screening Takashi et al. have identified a gene, called GEK1 at that time, essential
for the protection of plants from ethanol and acetaldehyde (Fujishige etal., 2004; Hirayama etal.,
2004). Later, using biochemical and bioinformatic analysis, GEK1 was identified to be a homolog
of archaeal D- aminoacyl- tRNA deacylase (DTD) (Wydau etal., 2007). DTDs are trans acting, chiral
proofreading enzymes involved in translation quality control and remove D- amino acids mischarged
onto tRNAs (Calendar and Berg, 1967; Soutourina et al., 1999; Soutourina et al., 2000; Kuncha
et al., 2019; Kumar et al., 2022). DTD function is conserved across all life forms where DTD1 is
present in bacteria and eukaryotes, DTD2 in land plants and archaea (Wydau etal., 2007; Ferri- Fioni
etal., 2006), and DTD3 in cyanobacteria (Wydau etal., 2009). All DTDs are shown to be important
in protecting organisms from D- amino acids (Wydau etal., 2007; Calendar and Berg, 1967; Sout-
ourina etal., 2000; Ferri- Fioni etal., 2006; Wydau etal., 2009). In addition, DTD2 was also found to
be involved in protecting plants against ethanol and acetaldehyde (Fujishige etal., 2004; Hirayama
etal., 2004; Wydau et al., 2007). Recently, we identified the biochemical role of archaea- derived
DTD2 gene in alleviating acetaldehyde stress in addition to resolving organellar incompatibility of
bacteria- derived DTD1 in land plants (Mazeed etal., 2021; Kumar et al., 2023). We have shown
that acetaldehyde irreversibly modifies D- aminoacyl- tRNAs (D- aa- tRNA) and only DTD2 can recycle
the modified D- aa- tRNAs thus replenishing the free tRNA pool for further translation (Mazeed etal.,
2021). Like acetaldehyde, elevated aldehyde spectrum (Figure1—figure supplement 1A) in plants
and archaea pose a threat to the translation machinery. The unique presence of DTD2 in organisms
with elevated aldehyde spectrum (plants and archaea) and its indispensable role in acetaldehyde
tolerance prompted us to investigate the role of archaeal DTD2 in safeguarding translation apparatus
of plants from various physiologically abundant toxic aldehydes.
Here, our in vivo and biochemical results suggest that formaldehyde and MG lead to toxicity in DTD2
mutant plants through D- aa- tRNA modification. Remarkably, out of all the aldehyde- modified D- aa-
tRNAs tested, only the physiologically abundant ones (i.e. D- aa- tRNAs modified by formaldehyde or
Source data 1. Biochemical data for the modication susceptibility of L- Ala- tRNAAla by multiple aldehydes and stability of formaldehyde- and MG-
modied and unmodied D- Tyr- tRNATyr substrates under alkaline conditions.
Source data 2. Table showing the expected and observed mass change upon aldehyde modication on D- Phe- tRNAPhe by electrospray ionisation mass
spectrometry (ESI- MS)/MS.
Figure supplement 1. Aldehydes modify the amino group of amino acids in D- aminoacyl- tRNAs (D- aa- tRNAs).
Figure 1 continued
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Figure 2. Elongation factor enantioselects aa- tRNAs through D- chiral rejection mechanism. (A)Surface representation showing the cocrystal structure
of elongation factor thermo unstable (EF- Tu) with L- Phe- tRNAPhe. Zoomed- in image showing the binding of L- phenylalanine with side chain projected
outside of binding site of EF- Tu (PDB id: 1TTT). (B)Zoomed- in image of amino acid binding site of EF- Tu bound with L- phenylalanine showing the
selection of amino group of amino acid through main chain atoms (PDB id: 1TTT). (C)Modelling of D- phenylalanine in the amino acid binding site of EF-
Tu shows severe clashes with main chain atoms of EF- Tu. Modelling of smallest chiral amino acid, alanine, in the amino acid binding site of EF- Tu shows
(D) no clashes with L- alanine and (E) clashes with D- alanine. (F)Modelling of D- alanine in the amino acid binding site of eEF- 1A shows clashes with main
chain atoms. (*Represents modelled molecule.) (G)Structure- based sequence alignment of elongation factor from bacteria, archaea, and eukaryotes
(both plants and animals) showing conserved amino acid binding site residues. (Key residues are marked with red star.)
The online version of this article includes the following gure supplement(s) for gure 2:
Figure supplement 1. Elongation factor protects L- aa- tRNAs from aldehyde modication.
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methylglyoxal) were deacylated by both archaeal and plant DTD2s. Therefore, plants have recruited
archaeal DTD2 as a potential detoxifier of all toxic aldehydes rather than only acetaldehyde as earlier
envisaged. Furthermore, DTD2 overexpressing Arabidopsis transgenic plants demonstrate enhanced
multi- aldehyde resistance that can be explored as a strategy for crop improvement.
Results
Aldehydes modify D-aa-tRNAs to disrupt protein synthesis
The presence of large amounts of chemically diverse aldehydes in plants and archaea (Figure1—
figure supplement 1A) encouraged us to investigate their influence on aa- tRNAs, a key component
of the translational machinery. We incubated aa- tRNAs with diverse aldehydes (from formaldehyde
[1- C] to decanal [10- C] including MG [3- C]) and investigated adduct formation with thin- layer chro-
matography (TLC) and electrospray ionisation mass spectrometry (ESI- MS). We observed that alde-
hydes modified aa- tRNAs irrespective of amino acid chirality (Figure1A–G and Figure 1—figure
supplement 1B). The mass change from formaldehyde, propionaldehyde, butyraldehyde, and MG
modification corresponds to a methyl, propyl, butyl, and acetonyl group, respectively (Figure1C–G).
Tandem fragmentation (MS2) of aldehyde- modified D- aa- tRNAs showed that all the aldehydes selec-
tively modify only the amino group of amino acids in D- aa- tRNAs (Figure 1—figure supplement
1C–G). Interestingly, upon a comparison of modification strength, the propensity of modification
decreased with increase in the aldehyde chain length with no detectable modification on decanal-
treated aa- tRNAs (Figure1A and H). The chemical reactivity of aldehyde is dictated by its electro-
philicity (LoPachin and Gavin, 2014). The electrophilicity of saturated aldehydes decreases with the
increasing chain length of aldehyde (LoPachin and Gavin, 2014; Pratihar, 2014), thereby reducing
modification propensity. Exceptionally, the modification propensity of MG is much higher than propi-
onaldehyde (Figure1H) which is also a three- carbon system (Figure1—figure supplement 1H) and it
is likely due to the high electrophilicity of the carbonyl carbon (LoPachin and Gavin, 2014). Also, the
aldehydes with higher propensity of modification are present in higher amounts in plants and archaea
(Figure1—figure supplement 1A). Further, we investigated the effect of aldehyde modification on
the stability of ester linkage of aa- tRNAs by treating them with alkaline conditions. Strikingly, even the
smallest aldehyde modification stabilised the ester linkage by~13- fold when compared with unmod-
ified aa- tRNA (Figure1I–J).
Elongation factor thermo unstable (EF- Tu) is shown to protect L- aa- tRNAs from acetaldehyde
modification (Mazeed etal., 2021). EF- Tu- based protection of L- aa- tRNAs can be extended to any
aldehydes with similar or bigger size than acetaldehyde but not formaldehyde. We sought to inves-
tigate the elongation factor- based protection against formaldehyde. To understand this, we have
done a thorough sequence and structural analysis. We analysed the aa- tRNA- bound elongation factor
structure from bacteria (PDB ids: 1TTT) and found that the side chain of amino acid in the amino
acid binding site of EF- Tu is projected outside (Figure2A and Figure2—figure supplement 1A).
In addition, the amino group of amino acid is tightly selected by the main chain atoms of elongation
factor thereby lacking a space for aldehydes to enter and then modify the L- aa- tRNAs and Gly- tRNAs
(Figure2B and Figure2—figure supplement 1B). Modelling of D- amino acid (either D- phenylalanine
or smallest chiral amino acid, D- alanine) in the same site shows serious clashes with main chain atoms
of EF- Tu, indicating a D- chiral rejection during aa- tRNA binding by elongation factor (Figure2C–E).
Next, we superimposed the tRNA- bound mammalian (from Oryctolagus cuniculus) eEF- 1A cryoEM
structure (PDB id: 5LZS) with bacterial structure to understand the structural differences in terms of
tRNA binding and found that elongation factor binds tRNA in a similar way (Figure2—figure supple-
ment 1C–D). Modelling of D- alanine in the amino acid binding site of eEF- 1A also shows serious
clashes with main chain atoms, indicating a general theme of D- chiral rejection during aa- tRNA binding
by elongation factor (Figure2F and Figure2—figure supplement 1E). Structure- based sequence
alignment of elongation factor from bacteria, archaea, and eukaryotes (both plants and mammals)
shows a strict conservation of amino acid binding site (Figure2G). Minor differences near the amino
acid side chain binding site (as indicated in Wolfson and Knight, FEBS Letters, 2005) might induce the
amino acid specific binding differences, if any (Figure2—figure supplement 1F). However, those
changes will have no influence when the D- chiral amino acid enters the pocket, as the whole side chain
would clash with the active site. To confirm these structural and sequence analyses biochemically,
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Figure 3. D- aminoacyl- tRNA deacylase2 (DTD2) acts as a general aldehyde detoxication system. Deacylation assays on formaldehyde-,
propionaldehyde-, methylglyoxal-, and butyraldehyde- modied D- Tyr- tRNATyr substrates by AtDTD2 (A–D),PhoDTD2 (E–H),AtDTD1 (I–L)(n=3).
(M)Table showing the effective activity concentration of AtDTD2, PhoDTD2, AtDTD1, archaeal peptidyl- tRNA hydrolase (PTH), and bacterial PTH that
completely deacylates aldehyde- modied D- Tyr- tRNATy r (‘-’ denotes no activity; *from Mazeed etal., 2021).
The online version of this article includes the following source data and gure supplement(s) for gure 3:
Source data 1. Biochemical data for deacylations of formaldehyde-, propionaldehyde-, MG-, and butyraldehyde- modied D- Tyr- tRNATyr substrates by
D- aminoacyl- tRNA deacylase1 (DTD1) and DTD2.
Source data 2. Biochemical data for deacylations of valeraldehyde- and isovaleraldehyde- modied D- Tyr- tRNATyr substrates by D- aminoacyl- tRNA
deacylase1 (DTD1) and DTD2.
Source data 3. Biochemical data for deacylations of formaldehyde-, propionaldehyde-, MG-, butyraldehyde-, valeraldehyde-, and isovaleraldehyde-
modied L- Tyr- tRNATy r substrates by D- aminoacyl- tRNA deacylase1 (DTD1), DTD2, peptidyl- tRNA hydrolase1 (PTH1) and PTH2.
Figure supplement 1. D- aminoacyl- tRNA deacylase2 (DTD2) is inactive on aldehyde- modied D- aminoacyl- tRNAs (D- aa- tRNAs) beyond three- carbon
aldehyde chain length.
Figure supplement 2. D- aminoacyl- tRNA deacylase2 (DTD2) acts as a general aldehyde detoxication system.
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bacterial EF- Tu (Thermus thermophilus) was used. EF- Tu was activated by exchanging the GDP with
GTP. Activated EF- Tu protected L- aa- tRNAs from RNase (Figure2—figure supplement 1G). Next,
we generated the ternary complex of activated EF- Tu and aa- tRNAs and incubated with formalde-
hyde. Reaction mixture was quenched at multiple time points and modification was assessed using
TLC. It has been seen that activated EF- Tu protected L- aa- tRNAs from smallest aldehyde suggesting
that EF- Tu is a dedicated protector of L- aa- tRNAs from all the cellular metabolites (Figure2—figure
supplement 1H). However, the lower affinity of D- aa- tRNAs with EF- Tu results in their modification
under aldehyde flux. Accumulation of these stable aldehyde- modified D- aa- tRNAs will deplete the
free tRNA pool for translation. Therefore, removal of aldehyde- modified D- aa- tRNAs is essential for
cell survival.
DTD2 recycles aldehyde-modified D-aa-tRNAs
Aldehyde- mediated modification on D- aa- tRNAs generated a variety of alkylated- D- aa- tRNA adducts
(Figure1A and Figure1—figure supplement 1B). While we earlier showed the ability of DTD2 to
remove acetaldehyde- induced modification, we wanted to test whether it can remove diverse range
of modifications ranging from smaller methyl to larger valeryl adducts to ensure uninterrupted protein
synthesis in plants. To test this, we cloned and purified Arabidopsis thaliana (At) DTD2 and performed
deacylation assays using different aldehyde- modified D- Tyr-(At)tRNATyr as substrates. DTD2 cleaved
majority of aldehyde- modified D- aa- tRNAs at 50 pM to 500 nM range (Figure 3A–D, Figure 3—
figure supplement 1A–B and Figure 3—figure supplement 2A–F). Interestingly, DTD2’s activity
decreases with increase in aldehyde chain lengths (Figure3A–D, Figure3—figure supplement 1A–B,
and Figure3—figure supplement 2A–F). To establish DTD2’s activity on various aldehyde- modified
D- aa- tRNAs as a universal phenomenon, we checked DTD2 activity from an archaeon (Pyrococcus
horikoshii [Pho]). DTD2 from archaea recycled short chain aldehyde- modified D- aa- tRNA adducts as
expected (Figure3E–G) and, like DTD2 from plants, it did not act on aldehyde- modified D- aa- tRNAs
longer than three carbons (Figure3H, Figure3—figure supplement 1C–D, and Figure3—figure
supplement 2G–L). Whereas the canonical chiral proofreader, DTD1, from plants was inactive on
all aldehyde- modified D- aa- tRNAs (Figure3I–L and Figure3—figure supplement 1E–F). Interest-
ingly, DTD2 was inactive on butyraldehyde, and higher chain length aldehyde- modified D- aa- tRNAs
(Figure3D and H, Figure3—figure supplement 1A–D, Figure3—figure supplement 2D–F, and
Figure3—figure supplement 2J–L). This suggests that DTD2 exerts its protection till propionalde-
hyde with a significant preference for methylglyoxal and formaldehyde- modified D- aa- tRNAs. It is
worth noting that the physiological levels of higher chain length aldehydes are comparatively much
lesser in plants and archaea (Figure1—figure supplement 1A), indicating the coevolution of DTD2
activity with the presence of toxic aldehydes. Even though both MG and propionaldehyde generate a
three- carbon chain modification, DTD2 showed~100- fold higher activity on MG- modified D- aa- tRNAs
(Figure3B–C, F–G, and M). It is interesting to note that peptidyl- tRNA hydrolase (PTH), which recy-
cles on N- acetyl/peptidyl- L- aa- tRNAs and has a similar fold to DTD2, was inactive on formaldehyde
and MG- modified L- and D- aa- tRNAs (Figure3M and Figure3—figure supplement 2M–Q). Overall,
our biochemical assays with multiple trans acting proofreaders (DTD1 and DTD2) and peptidyl- tRNA
recycling enzymes (both bacterial and archaeal PTH) suggest that DTD2 is the only aldehyde detoxi-
fier recycling the tRNA pool in both plants and archaea.
Absence of DTD2 renders plants susceptible to physiologically
abundant toxic aldehydes
Biochemical assays suggest that DTD2 may exert its protection for both formaldehyde and MG in
addition to acetaldehyde. To test this in vivo, we utilised an A. thaliana T- DNA insertion line (SAIL_288_
B09) having T- DNA in the first exon of DTD2 gene (Figure4A). We generated a homozygous line
(Figure4A) and checked them for ethanol sensitivity as ethanol metabolism produces acetaldehyde.
Similar to earlier results (Fujishige etal., 2004; Hirayama etal., 2004; Wydau etal., 2007), dtd2-/-
(dtd2 hereafter) plants were susceptible to ethanol (Figure4—figure supplement 1A) confirming the
non- functionality of DTD2 gene in dtd2 plants. We then subjected them to various concentrations
of formaldehyde and MG generally used for plant toxicity assays (Welchen etal., 2016; Wienstroer
et al., 2012; Achkor et al., 2003). These dtd2 plants were found to be sensitive to both formal-
dehyde and MG (Figure4B–G). This sensitivity was alleviated by complementing dtd2 mutant line
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Figure 4. D- aminoacyl- tRNA deacylase2 (DTD2) mutant plants are susceptible to physiologically abundant toxic aldehydes. (A)Schematics showing
the site of T- DNA insertion in (SAIL_288_B09) the rst exon of DTD2 gene and reverse transcriptase- polymerase chain reaction (RT- PCR) showing the
expression of DTD2 gene in wild type (Wt), dtd2-/-, dtd2-/-::AtDTD2 (rescue), and dtd2-/-::AtDTD2 H150A (catalytic mutant) plant 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 the
effect of (C) Murashige and Skoog agar (MSA), (D)1.5mM MG, (E)0.5mM D- Tyr and 1.5mM MG, (F)0.5mM formaldehyde, and (G) 0.5mM D- Tyr and
0.5mM 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). Ordinary one- way ANOVA test was used where p values higher than
0.05 are denoted as ns and p≤0.001 are denoted as ***. Graph showing the effect of (H) formaldehyde and (I) MG on germination of Wt, dtd2-/-, dtd2-/-
::AtDTD2 (rescue), and dtd2-/-::AtDTD2 H150A (catalytic mutant) plants (n=3).
The online version of this article includes the following source data and gure supplement(s) for gure 4:
Source data 1. Seedling surface area data and germination data for wild type (Wt), dtd2-/-, dtd2-/-::AtDTD2 (rescue), and dtd2-/-::AtDTD2 H150A
(catalytic mutant) plants under formaldehyde and MG with and without D- amino acid treatments.
Source data 2. Germination data for wild type (Wt), dtd2-/-, and dtd2-/-::AtDTD2 (rescue) plants under 1.5mM MG with 0.5mM D- tyrosine treatment.
Source data 3. Biochemical data for deacylations of acetaldehyde- modied D- Tyr- tRNATyr and L- Tyr- tRNATyr substrates by both wild type and catalytic
mutant D- aminoacyl- tRNA deacylase2 (DTD2) proteins.
Source data 4. Original les for reverse transcriptase- polymerase chain reaction (RT- PCR) analysis in Figure4A.
Figure supplement 1. MG and formaldehyde inhibit the germination of D- aminoacyl- tRNA deacylase2 (DTD2) mutant plants.
Figure supplement 2. Loss of D- aminoacyl- tRNA deacylase (DTD) results in accumulation of modied D- aminoacyl adducts on tRNAs in E. coli.
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with genomic copy of wild type DTD2 (Figure4A–G), indicating that DTD2- mediated detoxification
plays an important role in plant aldehyde stress. To further confirm the significance of DTD2 in plant
growth and development, we performed seed germination assays in dtd2 plants by evaluating the
emergence of radicle on third day post seed plating. As expected, dtd2 plants show a significant
reduction (~40%) in germination (Figure4H–I) and this effect was reversed in the DTD2 rescue line
(Figure4H–I). Interestingly, these toxic effects (on both growth and germination) of formaldehyde
and MG were enhanced upon D- amino acid supplementation (Figure4—figure supplement 1B).
These observations suggest that DTD2’s chiral proofreading activity is associated with aldehyde stress
removal activity as well. Moreover, to rule out the plausible role of any interacting partner or any other
indirect role of DTD2, we generated a catalytic mutant transgenic line containing a genomic copy of
AtDTD2 having H150A mutation (Ferri- Fioni etal., 2006; Figure4—figure supplement 1C–F). The
catalytic mutant line showed a similar phenotype as dtd2 plants under aldehyde stress (Figure4A–I),
confirming the role of DTD2’s biochemical activity in relieving general aldehyde toxicity in plants. We
tried to characterise the aldehyde- modified D- aminoacyl adducts on tRNAs with dtd2 mutant plants
extensively through northern blotting as well as mass spectrometry. However, due to the lack of infor-
mation about the tissue getting affected (root, shoot, etc.), identity of aa- tRNA, as well as location of
aa- tRNA (cytosol or organellar), we are so far unsuccessful in identifying them from plants. However,
we have used a bacterial surrogate system, Escherichia coli, as used earlier (Mazeed etal., 2021) to
show the accumulation of D- aa- tRNA adducts in the absence of DTD protein. We could identify the
accumulation of both formaldehyde- and MG- modified D- aa- tRNA adducts via mass spectrometry
(Figure4—figure supplement 2A–H). Overall, our results show that DTD2- mediated detoxification
protects plants from physiologically abundant toxic aldehydes.
Overexpression of DTD2 provides enhanced multi-aldehyde stress
tolerance to plants
Plants being sessile are constantly subjected to multiple environmental stresses that reduce agricul-
ture yield and constitute a serious danger to global food security (Zhu, 2016). Pyruvate decarboxylase
(PDC) transgenics are used to increase flood tolerance in plants but it produces~35- fold higher acet-
aldehyde than wild type plants (Bucher etal., 1994). Transgenics overexpressing enzymes known for
aldehyde detoxification like alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), alde-
hyde oxidase (AOX), and glyoxalase are shown to be multi- stress tolerant (Gupta etal., 2018; Zhao
etal., 2017; Nurbekova etal., 2021; Rodrigues etal., 2006). The sensitivity of dtd2 plants under
physiological aldehydes and biochemical activity of DTD2 prompted us to check if overexpression of
DTD2 can provide multi- aldehyde tolerance. We generated a DTD2 overexpression line with DTD2
cDNA cloned under a strong CaMV 35S promoter (Odell etal., 1985). We subjected the overexpres-
sion line, along with the wild type, to various aldehydes with or without D- amino acids. Strikingly, we
found that the DTD2 overexpression line was more tolerant to both the aldehydes (formaldehyde and
MG) when compared with wild type (Figure5A–C and Figure5—figure supplement 1A–C). DTD2
overexpression resulted in>50% increased seedling growth when compared with that of wild type
(Figure 5A and Figure5—figure supplement 1D). The growth difference was more pronounced
when D- amino acids were supplemented with varying concentrations of aldehydes (Figure5A and
Figure5—figure supplement 1D). Interestingly, DTD2 overexpression plants showed extensive root
growth under the influence of both formaldehyde and MG (Figure5—figure supplement 1A–C).
Plants produce these aldehydes in huge amounts under various stress conditions (Jardine etal.,
2009; Yadav etal., 2005) and plant tolerance to various abiotic stresses is strongly influenced by
root growth (Seo etal., 2020). The enhanced root growth by DTD2 overexpression under aldehyde
stress implies that DTD2 overexpression offers a viable method to generate multi- stress- resistant crop
varieties.
DTD2 appearance corroborates with the aldehyde burst in land plant
ancestors
After establishing the role of DTD2 as a general aldehyde detoxification system in the model land
plant system, we wondered if the multi- aldehyde detoxification potential of DTD2 was present in land
plant ancestors as well. Therefore, we checked the biochemical activity of DTD2 from a charophyte
algae, Klebsormedium nitens (Kn), and found that it also recycled aldehyde- modified D- aa- tRNAs
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adducts like other plant and archaeal DTD2s (Figure6A–C and Figure6—figure supplement 1A–C).
This suggests that the multi- aldehyde problem in plants has its roots in their distant ancestors, charo-
phytes. Next, we analysed the presence of other aldehyde metabolising enzymes across plants.
Multiple bioinformatic analyses have shown that land plants encode greater number of ALDH genes
compared to green algae (Tola etal., 2020; Islam and Ghosh, 2022) and glyoxalase family (GlyI, GlyII,
and GlyIII), known to clear MG, has expanded exclusively in streptophytic plants (Singla- Pareek etal.,
2020; Xu etal., 2023). We identified that land plants also encode greater number of AOX genes in
addition to ALDH genes compared to green algae (Figure6—figure supplement 1D). We delved
deeper into plant metabolism with an emphasis on formaldehyde and MG. A search for the formal-
dehyde (C00067) and MG (C00546) in KEGG database (Kanehisa etal., 2016) has shown that form-
aldehyde is involved in 5 pathways, 60 enzymes, and 94 KEGG reactions, while MG in 6 pathways, 16
Figure 5. Overexpression of D- aminoacyl- tRNA deacylase2 (DTD2) confers increased multi- aldehyde tolerance to A. thaliana. DTD2 overexpression
(OE) plants grow better than wild type Col- 0 under (A) 0.5mM, 0.75mM, 1.0mM, and 1.25mM of formaldehyde with and without 0.5mM D- tyrosine.
Cotyledon surface area (mm2) is plotted as parameter for seedling size (n=5–15). Ordinary one- way ANOVA test was used where p values higher 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 0.5mM, 0.75mM, 1.0mM, 1.25mM, 1.5mM
of MG and 0.5mM, 0.75mM, 1.0mM MG with 0.5mM D- tyrosine. (C)The quantitative polymerase chain reaction (qPCR) analysis showing fold change
of DTD2 gene expression in DTD2 OE plant line used (n=3).
The online version of this article includes the following source data and gure supplement(s) for gure 5:
Source data 1. Seedling surface area data for wild type (Wt) and D- aminoacyl- tRNA deacylase2 overexpression (DTD2 OE) plants under multiple
concentrations of formaldehyde with and without D- amino acid treatments.
Source data 2. The quantitative polymerase chain reaction (qPCR) analysis data of D- aminoacyl- tRNA deacylase2 (DTD2) gene expression in wild type
(Wt) and DTD2 overexpression (OE) plant line used.
Source data 3. Seedling surface area data for wild type (Wt) and D- aminoacyl- tRNA deacylase2 overexpression (DTD2 OE) plants under multiple
concentrations of formaldehyde with and without D- amino acid treatments.
Figure supplement 1. Overexpression of D- aminoacyl- tRNA deacylase2 (DTD2) confers multi- aldehyde tolerance with D- amino acid stress in A.
thaliana.
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enzymes, and 16 KEGG reactions. We did a thorough bioinformatic search for the presence of around
31 and 9 enzymes related to formaldehyde and MG, respectively, in KEGG database (Supplementary
file 1). Strikingly, we found that plants encode majority of the genes related for formaldehyde and MG
and they are conserved throughout land plants (Figure6D and Figure6—figure supplement 1E–G)
(Supplementary file 1). Plants produce significant amounts of formaldehyde while reshuffling pectin
in their cell wall during cell division, development, and tissue damage (Wu etal., 2018; Dorokhov
etal., 2018). Plants contain~33% pectin in their cell walls that provides strength and flexibility (Jarvis
etal., 1988). When checked for the presence of genes responsible for the pectin biosynthesis and
degradation, we identified that it is a land plant- specific adaptation that originated in early diverging
streptophytic algae (Figure6—figure supplement 1F) (Supplementary file 1). Overall, our bioin-
formatic analysis in addition to earlier studies has identified an expansion of aldehyde metabolising
repertoire in land plants and their ancestors indicating the sudden aldehyde burst accompanying
terrestrialisation which strongly correlates with the recruitment of DTD2 (Figure6E and Figure7).
Discussion
Plants produce more than 200,000 metabolites for crosstalk with other organisms (Kessler and Kalske,
2018). The burgeoning information on increased utilisation of aldehydes for signalling, defence, and
altering the ecological interactions with other organisms suggests their physiological importance in
plant life (Yadav etal., 2005). However, aldehydes are strong electrophiles that undergo addition
reactions with amines and thiol groups to form toxic adducts with biomolecules. Excessive aldehyde
accumulation irreversibly modifies nucleic acids and proteins resulting in cell death (Carlsson etal.,
2014; Pontel et al., 2015). In this work, we have shown that multiple aldehydes can cause toxicity
in dtd2 plants. Therefore, plants have recruited DTD2 as a detoxifier of aldehyde- induced toxicities
in the context of protein biosynthesis. Through this work, we find a correlation between physiolog-
ical abundance of various aldehydes, their modification propensity, and DTD2’s aldehyde protec-
tion range. Aldehydes with higher reactivity (formaldehyde, acetaldehyde, and MG) are present in
higher amounts in plants and archaea and DTD2 provides modified- D- aa- tRNA deacylase activity
against these aldehydes. DTD2’s biochemical activity decreases with increase in the aldehyde chain
length. Intriguingly, despite MG and propionaldehyde generating a three- carbon long modification,
DTD2 is ~100- fold more active on MG- modified D- aa- tRNAs. The absence of carbonyl carbon in
the propionaldehyde- modified substrate and DTD2’s preferential activity on the bulkier MG- modi-
fied substrate points to a clear evolutionary selection pressure for the abundant and physiologically
relevant aldehyde. In total contrast to DTD2, all PTH substrates contain carbonyl carbon at the alpha
position after the amino group of amino acid in L- aa- tRNA (Atherly, 1978). The inactivity of PTH
on MG- modified L- and D- aa- tRNAs suggests its specificity for carbonyl carbon at alpha position
(Figure3—figure supplement 2Q). Therefore, elucidating the structural basis for both enantioselec-
tion and modification specificity of DTD2 and PTH will throw light on these key mechanisms during
translation quality control.
The sensitivity of dtd2 plants to aldehydes of higher prevalence and hyper- propensity for modifi-
cation indicates the physiological coevolution of aldehyde phytochemistry and recruitment of DTD2
in land plants. Despite the toxic effects of reactive aldehydes, plants are being used as air purifiers as
they act as aldehyde scavengers from the environment (Teiri etal., 2018; Aydogan and Montoya,
2011; Wang etal., 2014; Li etal., 2016). Moreover, plants have higher removal rates for formalde-
hyde and acetaldehyde as compared to other higher chain length aldehydes from the environment
(Li etal., 2016). These aldehydes are produced under various biotic and abiotic stresses in plants
and overexpression of enzymes (PDC, ADH, ALDH, and glyoxalase) involved in aldehyde detoxifica-
tion are shown to provide multi- stress tolerance (Gupta etal., 2018; Quimio etal., 2000; Su etal.,
2020; Sun, 2019). In similar lines, here, we have also explored the possibility of DTD2 overexpression
in multi- aldehyde stress tolerance. Our in vivo results strongly suggests that DTD2 provides multi-
aldehyde stress tolerance in the context of detoxifying adducts formed on aa- tRNA. This facilitates
the release of free tRNA pool thus relieving translation arrest. DTD2 overexpression plants showed
extensive root growth as compared to wild type plants. Plant root growth is an indicator of multi- stress
tolerance (Seo etal., 2020). Therefore, our DTD2 overexpression approach could be explored further
in crop improvement strategies.
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Land Plants
Green Algae
Expansion of Aldehyde metabolising repertoire
(ALDH, AOX, Glyoxalase family)
Origin of Pectin in cell wall
and recruitment of DTD2
Figure 6. Terrestrialisation of plants is associated with expansion of aldehyde metabolising genes. Deacylation assays of KnDTD2 on (A) formaldehyde-,
(B)propionaldehyde-, and (C) MG- modied D- Tyr- tRNATyr (n=3). (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.
Figure 6 continued on next page
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The role of reactive aldehydes like formaldehyde in the origin of life is inevitable (Kitadai and
Maruyama, 2018). The presence of reactive aldehydes (Miller and Urey, 1959; Miller, 1957) and
D- amino acids (Parker etal., 2011; Naraoka etal., 2023) for such a long time suggests an ancient
origin of DTD2 activity in last archaeal common ancestor. As archaea thrive in extreme conditions,
they secrete enormous amount of formaldehyde into the environment as they grow (Moran etal.,
2016). We have shown that DTD2 from archaea can efficiently recycle physiologically abundant toxic
aldehyde- modified D- aa- tRNAs like plant DTD2s. The adduct removal activity was utilised by the
archaeal domain as they produce more aldehydes and thrive in harsh environments (Gribaldo and
Brochier- Armanet, 2006; Merino et al., 2019; Spang etal., 2017) and it was later acquired by
plants. Bog ecosystems, earlier proposed site for DTD2 gene transfer (Mazeed et al., 2021), are
highly anaerobic, rich in D- amino acids and ammonia (Taffner etal., 2018; Vranova et al., 2012;
Kharanzhevskaya et al., 2011), which lead to enhanced production of aldehydes (acetaldehyde
[Tadege and Kuhlemeier, 1997] and MG Borysiuk etal., 2018) in their inhabitants. Our bioinformatic
analysis in addition to earlier studies (Tola etal., 2020; Islam and Ghosh, 2022; Singla- Pareek etal.,
2020; Xu etal., 2023) has identified an expansion of aldehyde metabolising repertoire exclusively in
land plants and their ancestors indicating a sudden aldehyde burst associated with terrestrialisation.
Thus, recruitment of archaeal DTD2 by a land plant ancestor must have aided in the terrestrialisation
of early land plants. Considering the fact that there are no common incidences of archaeal gene
transfer to eukaryotes, it is unclear whether the DTD2 gene was transferred directly to land plant
ancestor from archaea or perhaps was mediated by an unidentified intermediate bacterium warrants
further investigation. Overall, the study has established the role of archaeal origin DTD2 in land plants
by mitigating the toxicity induced by aldehydes during protein biosynthesis.
Materials and methods
Plant material and growth conditions
Arabidopsis seeds of Columbia background were procured from the Arabidopsis Biological Resource
Center (Col- 0: CS28166; dtd2: SAIL_588_B09 [CS825029]). Plants were cultivated in a growth room
at 22°C with 16hr of light. Seeds were germinated on 1× Murashige- Skoog (MS) medium plates
containing 4.4g/l MS salts, 20g/l sucrose, and 8g/l tissue culture agar with pH 5.75 adjusted with
KOH at 22°C in a lighted incubator. Supplementary file 2 contains the primers used to genotype the
plants via polymerase chain reaction (PCR).
Construction of DTD2 rescue and DTD2 overexpression line
The coding sequence for Arabidopsis DTD2 (At2g03800) was PCR- amplified and inserted into
pENTR/D- TOPO for the overexpression line and genomic sequence for DTD2 (At2g03800) along with
its promoter (~2.4kb upstream region of DTD2 gene) was PCR- amplified and inserted into pENTR/D- -
TOPO for the rescue line (primer sequences available in Supplementary file 2). Site- directed muta-
genesis approach was used to create H150A (catalytic mutant) in plasmid used for rescue line. LR
Clonase II (Thermo Fisher Scientific) was used to recombine entry plasmids into (a) pH7FWG2 to
create the p35S::DTD2 line and (b) pZP222 to create rescue and catalytic mutant line. Agrobacterium
tumefaciens Agl1 was transformed with the above destination plasmids. The floral dip technique was
then used to transform Arabidopsis plants with a Columbia background (Clough and Bent, 1998).
The transgenic plants for overexpression were selected with 50µg/ml hygromycin and 1µg/ml Basta
(glufosinate ammonium) and rescue plant lines with 120µg/ml gentamycin and 1µg/ml Basta (glufos-
inate ammonium) supplemented with MS media.
The online version of this article includes the following source data and gure supplement(s) for gure 6:
Source data 1. Biochemical data for deacylations of formaldehyde-, propionaldehyde-, and MG- modied D- Tyr- tRNATyr substrates by KnDTD2.
Source data 2. Biochemical data for deacylations of butyraldehyde-, valeraldehyde-, and isovaleraldehyde- modied D- Tyr- tRNATyr substrates by
KnDTD2.
Figure supplement 1. Land plant evolution is associated with the expansion of aldehyde metabolising repertoire.
Figure 6 continued
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Aldehyde sensitivity assays and seedling size quantification
For aldehyde sensitivity assays, seeds were initially sterilised with sterilisation solution and plated on
1× MS medium agar plates containing varying concentrations of aldehydes with or without D- tyrosine.
Seeds were grown in a growth room at 22°C with 16hr of light. Plates were regularly observed and
germination percentage was calculated based on the emergence of radicle on third day post seed
plating. Phenotypes were documented 2weeks post germination and seedling size (n=4–15) was
quantified. For seedling size quantification imaging was done using Axiozoom stereo microscope with
ZEN 3.2 (blue edition) software and processed as necessary. Ordinary one- way ANOVA test was used
where p values higher than 0.05 are denoted as ns and p≤0.001 are denoted as ***.
Total RNA extraction and RT-qPCR
For the reverse transcriptase- quantitative polymerase chain reaction (RT- qPCR) experiment, seeds
were germinated and grown for 14days on MS plate and 200mg of seedlings were flash- frozen
in liquid nitrogen. The RNeasy Plant Minikit (QIAGEN) was used to extract total RNA according to
the manufacturer’s instructions. 4 μg of total RNA was used for cDNA synthesis with PrimeScript
first strand cDNA Synthesis Kit (Takara), according to the manufacturer’s instructions. The resultant
cDNA was diluted and used as a template for the RT- PCRs for DTD2 rescue and catalytic mutant lines
with EF- Tu (At1g07920) as the internal control. While qPCR was done to quantify the level of DTD2
Figure 7. D- aminoacyl- tRNA deacylase2 (DTD2) acts as a general aldehyde detoxier 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. These
aldehydes generate stable alkyl modication on D- aminoacyl- tRNA adducts and DTD2 is unique proofreader for these alkyl adducts. Therefore, DTD2
protects plants from aldehyde toxicity associated with translation apparatus emerged from expanded metabolic pathways and D- amino acids.
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overexpression for DTD2 overexpression line with appropriate primers (Supplementary file 2) and
Power SYBR Green PCR Master Mix (Thermo Fisher). Reactions were carried out in a Bio- Rad CFX384
thermocycler, with three technical replicates per reaction. The 2-ΔCq method was used for relative
mRNA levels calculation with actin (At2g37620) as the internal control. Prism 8 was used for graph
generation and statistical analysis.
Cloning, expression, and purification
DTD1 and DTD2 genes from A. thaliana (At) were PCR- amplified from cDNA, and DTD2 gene from K.
nitens (Kn) was custom synthesised, while DTD2 gene from P. horikoshii (Pho) and tyrosyl- tRNA synthe-
tase (TyrRS) of T. thermophilus (Tth) were PCR- amplified using their genomic DNA with primers listed
in Supplementary file 2. All the above- mentioned genes were then cloned into the pET28b vector
via restriction- free cloning (van den Ent and Löwe, 2006). E. coli BL21(DE3) was used to overexpress
all the above cloned genes except EcPheRS where E. coli M15 was used. As plant DTD2s, TyrRS,
and PheRS contained 6X His- tag, they were purified via Ni- NTA affinity chromatography, followed
by size exclusion chromatography (SEC) using a Superdex 75 column (GE Healthcare Life Sciences,
USA). Cation exchange chromatography was used to purify PhoDTD2 no- tag protein followed by SEC.
Purification method and buffers for all the purifications were used as described earlier (Ahmad etal.,
2013). All the purified proteins were stored in buffer containing 100mM Tris (pH 8.0), 200mM NaCl,
5mM 2- mercaptoethanol (β-ME), and 50% glycerol for further use.
Generation of α-32P-labelled aa-tRNAs
We have used A. thaliana (At) tRNAPhe, A. thaliana (At) tRNATyr , and E. coli (Ec) tRNAAla in this study.
All the tRNAs were in vitro transcribed using the MEGAshortscript T7 Transcription Kit (Thermo Fisher
Scientific, USA). tRNAs were then radiolabelled with [α-32P] ATP (BRIT- Jonaki, India) at 3’-end using
E. coli CCA- adding enzyme (Ledoux and Uhlenbeck, 2008). Aminoacylation of tRNAPhe, tRNATyr , and
tRNAAla with phenylalanine, tyrosine, and alanine respectively, were carried out as mentioned earlier
(Ahmad etal., 2013; Kuncha etal., 2018). TLC was used to quantify the aminoacylation as explained
(Mazeed etal., 2021).
Generation of adducts on aa-tRNAs for probing relative modification
propensity of aldehyde with aa-tRNA and substrate generation for
biochemical activity
A single- step method was used for probing relative modification propensity of the aldehyde with
aa- tRNA where 0.2µM of Ala- tRNAAla was incubated with different concentrations of aldehydes (2mM
and 10 mM) along with 20 mM NaCNBH3 (in 100 mM potassium acetate [pH 5.4]) as a reducing
agent at 37°C for 30min. The reaction mixture was digested with S1 nuclease and analysed on TLC.
Except for decanal, all the aldehydes modified Ala- tRNAAla. The method for processing and quan-
tification of modification on aa- tRNA utilised is discussed earlier (Mazeed etal., 2021). However,
a two- step method was used for generating substrates for biochemical assays as discussed earlier
(Mazeed etal., 2021). It was used to generate maximum homogenous modification on the aa- tRNAs
for deacylation assays. Briefly, 2µM aa- tRNAs were incubated with 20 mM of formaldehyde, and
methylglyoxal or 1M of propionaldehyde, butyraldehyde, valeraldehyde, and isolvaleraldehyde at
37°C for 30min. Samples were dried to evaporate excess aldehydes using Eppendorf 5305 Vacufuge
plus Concentrator. The dried mixture was then reduced with 20mM NaCNBH3 at 37°C for 30min. All
reactions were ethanol- precipitated at –30°C overnight or –80°C for 2hr. Ethanol precipitated pellets
were resuspended in 5mM sodium acetate (pH 5.4) and used for biochemical assays.
Deacylation assays
For biochemical activity assays, various enzymes like DTD1s, DTD2s, and PTHs were incubated with
different aldehyde modified and unmodified α-32P- labelled D- Tyr- tRNATyr substrates (0.2μM) in deac-
ylation buffer (20mM Tris pH 7.2, 5mM MgCl2, 5mM DTT, and 0.2mg/ml bovine serum albumin) at
37°C. An aliquot of 1µl of the reaction mixture was withdrawn at various time points and digested
with S1 nuclease prior to their quantification by TLC. The quantity of aldehyde- modified Tyr- AMP
at t=0min was considered as 100% and the the amount of modified Tyr- AMP at each time point
normalised with respect to t=0min was plotted. All biochemical experiments were repeated at least
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three times. The mean values of three independent observations were used to plot the graphs with
each error bar representing the standard deviation from the mean value.
Alkali treatment
Both aldehyde- modified and unmodified D- aa- tRNAs were digested with S1 nuclease before
subjecting to alkali treatment (for formaldehyde: 100nM S1- digested sample with 100mM Tris pH
9.0; for methylglyoxal: 100nM S1- digested sample with 200mM Tris pH 9.0) at 37°C. Alkali- treated
samples withdrawn at different time points were directly analysed with TLC. GraphPad Prism software
was used to calculate the half- life by fitting the data points onto the curve based on the first- order
exponential decay equation [St] = [S0]e- kt, where the substrate concentration at time t is denoted as [St
], [S0] is the concentration of the substrate at time 0, and k is the first- order decay constant.
Mass spectrometry
To identify the modification by various aldehydes on D- aa- tRNAs, modified and unmodified D- Phe-
tRNAPhe were digested with aqueous ammonia (25% of vol/vol NH4OH) at 70°C for 18 hr (Mazeed
etal., 2021). Hydrolysed samples were dried using Eppendorf 5305 Vacufuge plus Concentrator. Dried
samples were resuspended in 10% methanol and 1% acetic acid in water and analysed via ESI- based
mass spectrometry using a Q- Exactive mass spectrometer (Thermo Scientific) by infusing through
heated electrospray ionisation source operating at a positive voltage of 3.5 kV. Targeted selected ion
monitoring (t- SIM) was used to acquire the mass spectra (at a resolving power of 70,000@200m/z)
with an isolation window of 2m/z, i.e., theoretical m/z and MH+ ion species. The high energy collision-
induced MS- MS spectra with a normalised collision energy of 25 of the selected precursor ion species
specified in the inclusion list (having the observed m/z value from the earlier t- SIM analysis) were
acquired using the method of t- SIM- ddMS2 (at an isolation window of 1m/z at a ddMS2 resolving
power of 35,000@200m/z).
Characterisation of D-aa-tRNA adducts from E. coli
To identify the accumulation of D- aa- tRNA adducts, overnight grown primary culture of DTD1 knockout
E. coli was used to inoculate 1% secondary culture in minimal media with or without 2.5mM D- tyro-
sine. Secondary culture grown to OD650 (optical density at 650nm) 0.8 was subjected to respective
aldehyde treatment (0.01% final concentration) with 0.5mM NaCNBH3 at 37°C for 30min. Cultures
were pelleted and total RNA was isolated through acidic phenol chloroform method. Total RNA was
digested with three volumes of aqueous ammonia (25% of vol/vol NH4OH) at 70°C for 18 hr (Mazeed
et al., 2021). Hydrolysed samples were dried using Eppendorf 5305 Vacufuge plus Concentrator.
Dried samples were resuspended in 10% methanol and 1% acetic acid in water and analysed via ESI-
based mass spectrometry using a Q- Exactive mass spectrometer (Thermo Scientific) as mentioned
above.
Bioinformatic analysis
Protein sequences for various enzymes involved in formaldehyde and MG metabolism were searched
in KEGG GENOME database (http://www.genome.jp/kegg/genome.html) (RRID:SCR_012773)
through KEGG blast search and all blast hits were mapped on KEGG organisms to identify their taxo-
nomic distribution. KEGG database lacks genome information for charophyte algae so the presence
of desired enzymes in charophyte was identified by blast search in NCBI (https://www.ncbi.nlm.nih.
gov/) (RRID:SCR_006472). Protein sequences for elongation factor (both EF- Tu and eEF- 1a) for the
representative organisms were downloaded from NCBI through BLAST- based search. The structure-
based multiple sequence alignment of elongation factor was prepared using the T- coffee (http://
tcoffee.crg.cat/) (RRID:SCR_011818) server, and the sequence alignment figure was generated using
ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).
Structure models for elongation factor complexed with aa- tRNA were downloaded from RCSB- PDB
(https://www.rcsb.org/) and analysed with The PyMOL Molecular Graphics System, Version 2.0
Schrödinger, LLC. ‘ProteinInteractionViewer’ plugin for Pymol was used with default parameters to
identify and represent the molecular clashes in elongation factor structures with L- phenylalanine
and modelled D- phenylalanine, L- and D- alanine in the amino acid binding site of elongation factor.
Figures were prepared with The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.
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Quantification and statistical analysis
Quantification approaches and statistical analyses of the deacylation assays can be found in the rele-
vant sections of the Materials and methods section.
Acknowledgements
The authors acknowledge Dr. Mukesh Lodha, CSIR- CCMB, for fruitful discussions and Gokulan CG,
CSIR- CCMB, for qRT- PCR- related help. PK and SJM thank CSIR, India, for Research Fellowship. RS
acknowledges healthcare theme project (MLP- 0162, MLP- 0138), CSIR, India, JC Bose Fellowship of
SERB, India, and Centre of Excellence Project (GAP- 0473) of Department of Biotechnology, India.
Additional information
Competing interests
Rajan Sankaranarayanan: Reviewing editor, eLife. The other authors declare that no competing inter-
ests exist.
Funding
Funder Grant reference number Author
Council of Scientic and
Industrial Research, India
Research Fellowship Pradeep Kumar
Shivapura Jagadeesha
Mukul
Council of Scientic and
Industrial Research, India
MLP-0138 Rajan Sankaranarayanan
Council of Scientic and
Industrial Research, India
MLP-0162 Rajan Sankaranarayanan
Science and Engineering
Research Board
J.C. Bose Fellowship Rajan Sankaranarayanan
Department of
Biotechnology, Ministry of
Science and Technology,
India
GAP-0473 Rajan Sankaranarayanan
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Pradeep Kumar, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visual-
ization, Methodology, Writing – original draft, Writing – review and editing; Ankit Roy, Data curation,
Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing; Shivapura
Jagadeesha Mukul, Investigation, Visualization, Methodology, Writing – review and editing; Avinash
Kumar Singh, Resources, Investigation, Methodology, Writing – review and editing; Dipesh Kumar
Singh, Resources, Methodology, Writing – review and editing; Aswan Nalli, Resources, Investigation,
Writing – review and editing; Pujaita Banerjee, Kandhalu Sagadevan Dinesh Babu, Investigation,
Writing – review and editing; Bakthisaran Raman, Investigation, Methodology, Writing – review and
editing; Shobha P Kruparani, Imran Siddiqi, Resources, Data curation, Methodology, Writing – review
and editing; Rajan Sankaranarayanan, Conceptualization, Resources, Data curation, Formal analysis,
Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft,
Project administration, Writing – review and editing
Author ORCIDs
Pradeep Kumar
https://orcid.org/0000-0001-8335-5816
Ankit Roy
http://orcid.org/0000-0003-3202-9230
Avinash Kumar Singh
http://orcid.org/0000-0001-6572-6732
Kandhalu Sagadevan Dinesh Babu
http://orcid.org/0000-0002-1491-1433
Research article Biochemistry and Chemical Biology | Plant Biology
Kumar etal. eLife 2023;13:RP92827. DOI: https://doi.org/10.7554/eLife.92827 18 of 22
Shobha P Kruparani
https://orcid.org/0000-0002-8955-1647
Rajan Sankaranarayanan
https://orcid.org/0000-0003-4524-9953
Peer review material
Reviewer #1 (Public Review): https://doi.org/10.7554/eLife.92827.3.sa1
Reviewer #2 (Public Review): https://doi.org/10.7554/eLife.92827.3.sa2
Author Response https://doi.org/10.7554/eLife.92827.3.sa3
Additional files
Supplementary files
• Supplementary file 1. Presence of enzymes related to formaldehyde, MG, and pectin in all KEGG
organisms.
• Supplementary file 2. List of DNA primers used in the study.
• MDAR checklist
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files;
source data files have been provided for Figures1, 3–6 and associated figure supplements.
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