Archaeal origin translation proofreader imparts multialdehyde stress tolerance to land plants

Reviewer #1 (Public Review):

Summary:
This work is an extension of the authors' earlier work published in Sci Adv in 2001, wherein the authors showed that DTD2 deacylates N-ethyl-D-aminoacyl-tRNAs arising from acetaldehyde toxicity. The authors in this study, investigate the role of archaeal/plant DTD2 in the deacylation/detoxification of D-Tyr-tRNATyr modified by multiple other aldehydes and methylglyoxal (produced by plants). Importantly, the authors take their biochemical observations to plants, to show that deletion of DTD2 gene from a model plant (Arabidopsis thaliana) makes them sensitive to the aldehyde supplementation in the media especially in the presence of D-Tyr. These conclusions are further supported by the observation that the model plant shows increased tolerance to the aldehyde stress when DTD2 is overproduced from the CaMV 35S promoter. The authors propose a model for the role of DTD2 in the evolution of land plants. Finally, the authors suggest that the transgenic crops carrying DTD2 may offer a strategy for stress-tolerant crop development. Overall, the authors present a convincing story, and the data are supportive of the central theme of the story.

Strengths:
Data are novel and they provide a new perspective on the role of DTD2, and propose possible use of the DTD2 lines in crop improvement.

Weaknesses:
(a) Data obtained from a single aminoacyl-tRNA (D-Tyr-tRNATyr) have been generalized to imply that what is relevant to this model substrate is true for all other D-aa-tRNAs (term modified aa-tRNAs has been used synonymously with the modified Tyr-tRNATyr). This is not a risk-free extrapolation. For example, the authors see that DTD2 removes modified D-Tyr from tRNATyr in a chain-length dependent manner of the modifier. Why do the authors believe that the length of the amino acid side chain will not matter in the activity of DTD2?
(b) While the use of EFTu supports that the ternary complex formation by the elongation factor can resist modifications of L-Tyr-tRNATyr by the aldehydes or other agents, in the context of the present work on the role of DTD2 in plants, one would want to see the data using eEF1alpha. This is particularly relevant because there are likely to be differences in the way EFTu and eEF1alpha may protect aminoacyl-tRNAs (for example see description in the latter half of the article by Wolfson and Knight 2005, FEBS Letters 579, 3467-3472).

Reviewer #2 (Public Review):

In bacteria and mammals, metabolically generated aldehydes become toxic at high concentrations because they irreversibly modify the free amino group of various essential biological macromolecules. However, these aldehydes can be present in extremely high amounts in archaea and plants without causing major toxic side effects. This fact suggests that archaea and plants have evolved specialized mechanisms to prevent the harmful effects of aldehyde accumulation.

In this study, the authors show that the plant enzyme DTD2, originating from archaea, functions as a D-aminoacyl-tRNA deacylase. This enzyme effectively removes stable D-aminoacyl adducts from tRNAs, enabling these molecules to be recycled for translation. Furthermore, they demonstrate that DTD2 serves as a broad detoxifier for various aldehydes in vivo, extending its function beyond acetaldehyde, as previously believed. Notably, the absence of DTD2 makes plants more susceptible to reactive aldehydes, while its overexpression offers protection against them. These findings underscore the physiological significance of this enzyme.

Author Response

We are grateful to the reviewers for their positive feedback with their comments and suggestions on the manuscript. Reviewer 1 has indicated two weaknesses and Reviewer 2 has none. With this provisional reply, we address the two concerns of the Reviewer 1:

1. Data obtained from a single aminoacyl-tRNA (D-Tyr-tRNATyr) have been generalized to imply that what is relevant to this model substrate is true for all other D-aa-tRNAs. This is not a risk-free extrapolation. Why do the authors believe that the length of the amino acid side chain will not matter in the activity of DTD2?

We thank the reviewer for bringing up this important point. We wish to clarify that only a few of the aminoacyl-tRNA synthetases are known to charge D-amino acids and only D-Leu (Yeast), D-Asp (Bacteria, Yeast), D-Tyr (Bacteria, Cyanobacteria, Yeast) and D-Trp (Bacteria) show toxicity in vivo in the absence of known DTD (Soutourina J. et al., JBC, 2000; Soutourina O. et al., JBC, 2004; Wydau S. et al., JBC, 2009). D-Tyr-tRNATyr is used as a model substrate to test the DTD activity in the field because of the conserved toxicity of D-Tyr in various organisms. DTD2 has been shown to recycle D-Asp-tRNAAsp and D-Tyr-tRNATyr with the same efficiency both in vitro and in vivo (Wydau S. et al., NAR, 2007). Moreover, we have previously shown that it recycles acetaldehyde-modified D-Phe-tRNAPhe and D-Tyr-tRNATyr in vitro (Mazeed M. et al., Science Advances, 2021). We have earlier shown that DTD1, another conserved chiral proofreader across bacteria and eukaryotes, acts via a side chain independent mechanism (Ahmad S. et al., eLife, 2013). Considering the action on multiple side chains with different chemistry and size, it can be proposed with reasonable confidence that DTD2 also operates based on a side chain independent manner.

1. While the use of EFTu supports that the ternary complex formation by the elongation factor can resist modifications of L-Tyr-tRNATyr by the aldehydes or other agents, in the context of the present work on the role of DTD2 in plants, one would want to see the data using eEF1alpha. This is particularly relevant because there are likely to be differences in the way EFTu and eEF1alpha may protect aminoacyl-tRNAs (for example see description in the latter half of the article by Wolfson and Knight 2005, FEBS Letters 579, 3467-3472).

We thank the reviewer for bringing another important point. We analysed the aa-tRNA bound elongation factor structures from both bacteria (PDB id: 1TTT) and mammal (PDB id: 5LZS) and found that the amino acid binding site is highly conserved where side chain of amino acid is projected outside. Modelling of D-amino acid in the same site shows serious clashes, indicating D-chiral rejection during aa-tRNA binding by elongation factor. 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. 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. 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. We will present a sequence and structural conservation analysis to clarify this important point in our revised manuscript. Overall, our structural analysis suggests a conserved mode of aa-tRNA selection by elongation factor across life forms and therefore, our biochemical results with bacterial elongation factor Tu (EF-Tu) reflect the protective role of elongation factor in general across species.

In our revised manuscript, we will provide a thorough point-by-point response to the above as well as all the specific reviewer comments. We also intend to include new analysis with updated data that would address the key questions raised by the reviewers.