Arabidopsis TRM5 encodes a nuclear-localised bifunctional tRNA guanine and inosine-N1-methyltransferase that is important for growth

Modified nucleosides in tRNAs are critical for protein translation. N1-methylguanosine-37 and N1-methylinosine-37 in tRNAs, both located at the 3’-adjacent to the anticodon, are formed by Trm5 and here we describe Arabidopsis thaliana AtTrm5 (At3g56120) as a Trm5 ortholog. We show that AtTrm5 complements the yeast trm5 mutant, and in vitro methylates tRNA guanosine-37 to produce N1-methylguanosine (m1G). We also show in vitro that AtTRM5 methylates tRNA inosine-37 to produce N1-methylinosine (m1I) and in Attrm5 mutant plants, we show a reduction of both N1-methylguanosine and N1-methylinosine. We also show that AtTRM5 is localized to the nucleus in plant cells. Attrm5 mutant plants have overall slower growth as observed by slower leaf initiation rate, delayed flowering and reduced primary root length. In Attrm5 mutants, mRNAs of flowering time genes are less abundant and correlated with delayed flowering. Finally, proteomics data show that photosynthetic protein abundance is affected in mutant plants. Our findings highlight the bifunctionality of AtTRM5 and the importance of the post-transcriptional tRNA modifications m1G and m1I at tRNA position 37 in general plant growth and development.


Introduction
RNA has over 100 different post-transcriptional modifications that have been identified in organisms across all three domains of life (1)(2)(3)(4)(5). While several RNA modifications have been recently identified on mRNAs in plants and animals, tRNAs are still thought to be the most extensively modified cellular RNAs (6)(7)(8)(9). These tRNA modifications are introduced at the post-transcriptional level by specific enzymes. These enzymes recognize polynucleotide substrates and modify individual nucleotide residues at highly specific sites. Some tRNA modifications have been shown to have a clear biological and molecular function (10,11). Several tRNA modifications around the anticodon have been demonstrated to have crucial functions in translation, for example, by enhancing decoding (12), influencing the propensity to ribosomal frameshifting or facilitating wobbling (13)(14)(15). Modifications distal to the tRNA anticodon loop can also directly influence the tRNA recognition and/or translation process (16) or can have roles in tRNA folding and stability (1,17). However, the precise functions of many tRNA modifications still remain unknown despite often being conserved across species. Often, loss of a tRNA modification does not negatively impair cell growth or cell viability under standard laboratory growth conditions (18).
However, under environmental stress, such mutants display a discernible phenotype (10).
The tRNA anticodon loop position 37 is important to maintain translational fidelity and efficiency (11,19) and almost all tRNAs are modified at this site. In bacteria, N 1methylation of guanosine at position 37, m 1 G37, is performed by TrmD-type enzymes (20). In Archaea and Eukaryotes, the m 1 G37 modification is enzymatically performed by functionally and evolutionarily unrelated Trm5-type proteins (21). In yeast Saccharomyces cerevisiae, m 1 G37 methylation of cytoplasmic and mitochondrial tRNAs is performed by Trm5p and complete loss of function mutants are lethal (22,23). In humans, TRMT5 (tRNA methyltransferase 5), catalyzes the formation of m 1 G37 in vivo, and mitochondrial tRNA PRO and tRNA LEU have been found to also contain m 1 G37 (24,25). Mutations in human TRMT5 cause patients to have multiple respiratory-chain deficiencies and a reduction in mitochondrial tRNA m 1 G37 (11). In plants, the enzymes catalysing m 1 G37 methylation have not been identified to date. N 1 -methylguanosine has been described in eukaryotic tRNAs at two positions; at position 37 catalysed by Trm5, and the other at position 9 catalysed by Trm10 (26).
Trm5 in humans, yeast, and Pyrococcus abyssi has been described as having multifunctionality (23,24,27). In contrast to TrmD which requires a guanosine at position 37, human Trm5 can also recognise and methylate inosine at position 37 with some limited activity (24). Similarly,Trm5p have also been shown to catalyse inosine to m 1 I modification in yeast in a two-step reaction, where the first adenosine-to-inosine modification was mediated by Tad1p (10,18,22,28). As m 1 G is an intermediate during the modification of guanosine to wybotusine (yW), tRNAs from trm5 mutants were also devoid of yW (22). The yeast Trm5p protein has been shown to be localised to the cytoplasm and mitochondria (23) and it is thought that Trm5p protein present in the mitochondria is required to prevent unmodified tRNA affecting translational frameshifting (22). In the unicellular parasite Trypanosome brucei, Trm5 was located in both the nucleus and mitochondria and reducing Trm5 expression led to reduced mitochondria biogenesis and impaired growth (29). Interestingly, Trm5 and m 1 G37 were shown to be essential for mitochondrial protein synthesis but not cytosolic translation (29).
Little is known about tRNA modifying enzymes in plants. Here, we report the identification and functional analysis of AtTrm5 (At3g56120) from the model plant Arabidopsis thaliana. We demonstrate that Attrm5 mutants are slower growing, have reduced shoot and root biomass and display late flowering. Furthermore, we demonstrate that in vitro TRM5 is required for m 1 G37 and m 1 I37 methylation at the position 3' to the anticodon and in vivo tRNAs enriched from Attrm5 plants have reduced m 1 G and m 1 I.

Plant material and root growth experiments
Arabidopsis thaliana (Columbia accession) wild type and mutant plants were grown in Phoenix Biosystems growth under metal halide lights as previously described (30).
Analysis of root phenotypes was carried out on 11-day-old seedlings grown on ½ MS agar plates. A flatbed scanner (Epson) was used to non-destructively acquire images of seedling roots grown on the agar surface. Once captured, the images were analyzed by software package RootNav (33,34).

Plasmid construction and generation of transgenic plants
For the 35SCaMV:TRM5 construct, the full-length genomic region of At3g56120 including the 5'UTR and 3'UTR was amplified from Col-0 genomic DNA template with primers provided in (Supplementary Table S1) and cloned into Gateway entry vector pENTRTM/SD/D-TOPO (Invitrogen). The insert was sequenced and then cloned into the binary destination vector pGWB5 by an LR recombination reaction, using the

Sub-cellular localization of TRM5
For analysis of subcellular localization of TRM5, the construct described above was introduced into A. tumefaciens strain GV3101 and transiently expressed in 5-week-old

Quantitative RT-PCR
For the transcription profiling of flowering-related genes and circadian clock-related genes, 17-day-old seedlings were sampled from Zeitgeber time (ZT) 1 and collected every 3h during the day and night cycles, respectively. Total RNA was extracted the leaf samples using Trizol reagent (Invitrogen). The relative expression levels of AtTRM5 were determined using quantitative real-time PCR with gene-specific primers (Supplementary Table S1). Real-time PCR was performed using the StepOnePlus real-time PCR system (Applied Biosystems) using Absolute SYBR Green ROX mix (Applied Biosystems) for quantitation. Three biological replicates were carried out for each sample set. The relative expression was corrected using a reference gene EF1alpha (At5g60390) and calculated using the 2 -∆∆Ct method as described previously (10).

mRNA-sequencing
Total RNA, 1 ug, was extracted from 20-day-old Arabidopsis leaf samples using Trizol reagent (Invitrogen) and purified using the RNAeasy Mini RNA kit (Qiagen). One hundred nanograms of RNA were used for RNA-seq library construction according the manufacturer's recommendations (Illumina). First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen). After second strand cDNA synthesis and adaptor ligation, cDNA fragments were enriched, purified and then sequenced on the Illumina Hiseq X Ten. Three biological replicates were used for RNA-seq experiments.

tRNA purification and tRNA-sequencing
Total RNA was isolated from wild type and trm5 10-day-old Arabidopsis seedlings using the Spectrum Plant total RNA kit (SIGMA-ALDRICH) and contaminating DNA removed using DNase I (SIGMA-ALDRICH). To enrich for tRNAs, 10μg of total RNA was separated on a 10% polyacrylamide gel, the region containing 65-85 nts was removed and RNA was purified as previously described (31). Purified tRNAs were used for library construction using NEB Ultradirectional RNA library kit. Given the short sequences of tRNAs, the fragmentation step of the library preparation was omitted,

Yeast complementation
AtTrm5 (At3g56120) and ScTrm5 (YHR070W) was PCR amplified from cDNA and cloned into pYE19 using a Gibson assembly reaction (NEB). Mutant AtTrm5 (R166D) was generated by synthesising gene blocks (IDT) with nucleotides that mutated the translated proteins at R166 and the gene block was cloned into pYE19 using a Gibson assembly reaction (NEB). Yeast △trm5 (Mat a, hisD1, leu2D1, met15D0, trm5:KanMX) was previously described (37). Recombinant plasmids were transformed into △trm5 mutant strain and the resulting strain was analysed for growth phenotypes and m 1 G nucleoside levels.
Proportion estimation: The common SNPs identified in wild type and trm5-1 samples were cross compared. SNPs at position 37 were extracted and analysed using vcftools (47). Changes in base pair modifications were indicated by base substitution due to the property of next generation sequencing as mentioned in (11,48). The ratio of the expected A-to-T conversion in wild type samples and both the ratio of A-to-T (indicating no change in comparison to wild type) and A-to-G in trm5-1 were analyzed as an indication of m 1 I depletion. Identification of other base changes was attempted to identify putative m 1 I modification.

Sequence analysis of tRNA editing
tRNA purification and tRNA editing analysis were performed as described previously (10). Cytosolic tRNA-Ala (AGC) were amplified by reverse transcriptase (RT)-PCR with specific primers (tRNA-Ala-f and tRNA-Ala-r, Supplementary Table S1), and purified PCR products were directly Sanger sequenced.

TMT-based proteome determination and data analysis
Total protein was extracted from 20-day-old Arabidopsis leaf samples and purified according to a method described by (49). Protein digestion was performed according to FASP procedure described previously (50), and 100 ųg peptide mixture of each

Identification of At3g56120 as a TRM5 homolog
In yeast (Saccharomyces cerevisiae), m 1 G37 nucleoside modification is catalysed by ScTrm5 (22). We searched for Arabidopsis thaliana homologs by using blastp and HMMER and identified a high confidence candidate, At3g56120, with 49% similarity to ScTrm5 (Supplementary Figure S1). Alignment of At3g56120 with yeast, human, Drosophilia, Pyrocococcus, and Methanococcus Trm5 homologs identified three conserved motifs and catalytically required amino acids (R166, D192, E206) present in At3g56120 (Supplementary Figure S1). We subsequently will refer to At3g56120 as AtTrm5. In the Arabidopsis genome, AtTrm5 has homology to At4g27340 and At4g04670 and both genes were recently been named as Trm5B and Trm5C respectively (Supplementary Figure S1 and (51)). We have also identified Trm5 homologs in algae, bryophytes and vascular plants ( Figure 1A).
To functionally characterize AtTrm5 we isolated two T-DNA insertions, SALK_022617 and SALK_032376, and identified homozygous mutant plants for each insertion ( Figure 1B). SALK_022617 and SALK_032376 were named trm5-1 and trm5-2, respectively. Next, we measured AtTrm5 mRNA abundance in both mutants and detected almost no transcripts in both mutants ( Figure 1C). We generated a genomic construct of AtTrm5 that contained the endogenous promoter, coding region and UTRs, transformed the construct into trm5-2 and demonstrated that the AtTrm5 mRNA levels were similar in two complemented lines when compared to wild type plants.
Subsequently, the extracted tRNAs from wild type and the trm5 mutants were purified, digested and modified nucleosides measured by mass spectrometry ( Figure 1D). In both trm5 mutant alleles, nucleoside m 1 G levels were reduced to about 30% of the wild type and m 1 G levels were restored to wild type levels in both complemented lines ( Figure 1D). Nucleoside m 1 G is present at tRNA positions 9 and 37 (22), therefore the residual m 1 G levels in both trm5 mutants may be the result of tRNA m 1 G at position 9.
In yeast, Trm5 has also been reported to also catalyse m 1 I on tRNAs (10,22,51). We therefore measured m 1 I levels in purified tRNAs from both Arabidopsis trm5 mutants and wild type control plants. In both trm5-1 and trm5-2 mutant alleles, nucleoside m 1 I levels were reduced to about 10% of wild type levels and were restored to wild type levels in plants of both complemented lines ( Figure 1D). In summary, in Arabidopsis thaliana we identified At3g56120 as a Trm5 homolog in Arabidopsis thaliana, identified two AtTrm5 mutant alleles, trm5-1 and trm5-2, and both mutants showed a significant reduction in m 1 G and m 1 I.

AtTRM5 m 1 G methyltransferase activity
To test AtTrm5 m 1 G methyltransferase activity in vivo, a yeast, a trm5 mutant strain in yeast (Saccharomyces cerevisiae, Sc) that is defective for the tRNA m 1 G37 modification was used for genetic complementation. The mutant not only has defective tRNA m 1 G37 but also a slow growth phenotype when compared to wild type or a congenic strain (Figure 2A). Full-length ScTrm5 and AtTrm5 were cloned into yeast expression vectors. From the AtTrm5 expression vector, a catalytically inactive mutant Attrm5 R166D was generated by site-directed mutagenesis. After the three vectors had been transformed into the yeast trm5 mutant, cell growth and m 1 G nucleoside levels were observed ( Figure 2). Not only were the slow growth and nucleoside levels rescued when expressing ScTrm5 but they were also rescued when expressing AtTrm5 (Figure 2A and B). However, the catalytically inactive Attrm5 R166D did not rescue either the slow growth or m 1 G nucleoside levels (Figure 2A and B).
To test the m 1 G methyltransferase activity of AtTRM5 in vitro, we incubated purified recombinant proteins with tRNA substrates and measured the m 1 G levels. We  Figure 2D). m 1 G was detected only when AtTRM5 was provided ( Figure   2D) and in a dosage dependent manner. No m 1 G was detected when the catalytically inactive mutant AtTRM5 was provided ( Figure 2D). To test the specificity of the methyltransferase activity on tRNA-Asp guanine at position 37, the guanine nucleotide was mutated to an adenine nucleotide (tRNA-Asp-A37) and the m 1 G methyltransferase activity was measured. No m 1 G was detected after incubation with the fusion proteins ( Figure 2D). The overall results of the yeast complementation experiments suggest that guanosine methylation occurred at position 37 of tRNA.

AtTRM5 tRNA m 1 I methyltransferase activity
Previously in plants, TAD1 was demonstrated to oxidatively deaminate adenosine at position 37 of tRNA-Ala-(UGC) to inosine, and subsequently methylated by an unknown enzyme to N1-methylinosine (m1I; Figure 3A). Human TRM5 has been reported to methylate tRNA I37 but with limited activity (24). Given our observation that Attrm5 mutant plants had reduced m 1 I ( Figure 1D), we asked the question whether AtTRM5 has methyltransferase activity on tRNA I37. We developed a two-step approach, whereby purified AtTAD1 was first incubated with the substrate tRNA-Ala-A37 to produce tRNA-Ala-I37 and then the inosine methyltransferase activity of AtTRM5 was measured by incubating AtTRM5 with the tRNA-Ala-I37 substrate.
Previously, in yeast ScTAD1 was demonstrated to deaminate tRNA-Ala-A37 to tRNA-Ala-I37 in vitro (22) and Arabidopsis thaliana tad1 mutants were reported to have reduced tRNA-Ala-I37 (10). We expressed AtTAD1 as a GST fusion protein and  Figure 3B). m 1 I was detected only when GST-AtTAD1 and GST-AtTRM5 were provided, and its production occurred in a dosage-dependent manner ( Figure 3B). No m 1 I was detected when the catalytically inactive mutants AtTAD1 E76S or AtTRM5 R166D were provided ( Figure   3B). To test the specificity of the methyltransferase activity on tRNA-Ala alanine at position 37, the alanine nucleotide was mutated to a cytosine nucleotide (tRNA-Ala-C37) and the m 1 I methyltransferase activity was measured. No m 1 I was detected after incubation with the fusion proteins ( Figure 3B). Collectively, these findings interestingly suggest that inosine methylation also occurrs at position 37, in addition to guanosine methylation.

13
To test the AtTRM5 inosine methyltransferase activity in vivo, we measured tRNA position modifications by cDNA sequencing from either mutant or wild type plants ( Figure 3C and D). In the sequencing assay, modification events at position 37 of tRNAs can be directly detected by sequencing of amplified cDNA obtained by reverse transcription and comparison to the DNA reference sequence as inosine is read as guanine (G) and m 1 I is read as thymine (T) by the reverse transcriptase (11,48). As AtTRM5 methylates tRNA-Ala-I37 to tRNA-Ala-m 1 I37. We confirmed this pathway in tad1 and trm5 mutant plants by Sanger sequencing of tRNA-Ala-AGC ( Figure 3D). As expected, at position 37, A was substituted to T in the wild type, whereas in trm5 mutants a G and in tad1 mutants an A were observed ( Figure 3D). These sequencing results are consistent with AtTAD1 first deaminating A37 to I37 as previously reported by (10), and AtTRM5 then methylating I37 to m 1 I. We also attempted to detect the putative loss of m 1 G in the tRNA-sequencing data, as it has been reported that m 1 G is prone to be called as a T in sequencing (48). However, this was not observed in our datasets. Together, our in vitro and in vivo data provide support for AtTRM5 possessing tRNA m 1 I methyltransferase activity.

AtTRM5 is localized to the nucleus
In yeast, ScTRM5 is localized to both the nucleus and mitochondria (23,53).
Localisation to mitochondria is thought to be important as yeast strains with only nuclear-localized ScTRM5 exhibited a significantly lower rate of oxygen consumption (23). In order to determine to which subcellular compartment(s) AtTRM5 is localized in Nicotiana benthamiana, we fused TRM5 to the Green Fluorescent Protein (GFP) reporter, transiently infiltrated the construct into leaves and performed laser-scanning confocal microscopy to detect GFP fluorescence. To unambiguously identify the nucleus, we stained the cells with DAPI. When we imaged the cells (n=100), we observed distinct DAPI fluorescence in a single large circular structure per cell, as expected for the nucleus (Figure 4). Next, we imaged the same cells for GFP fluorescence ( Figure 4C) and overlayed the DAPI and GFP fluorescence. The two fluorescence signals showed perfect overlap ( Figure 4D). We then searched for nuclear localisation signals (NLS) using the LOCALIZER (http://localizer.csiro.au/),  (22), which explains the outcome from LOCALIZER and LocSigDB. In summary, we conclude that, unlike in yeast, AtTRM5 in Arabidopsis is only localized to the nucleus and may be imported from the cytoplasm into the nucleus by the importin ⍺-dependent pathway.

Proteins involved in photosynthesis are affected in trm5 mutant plants
Next, we performed a proteomic analysis to identify proteins that differentially  Table S2). 102 proteins were upregulated, and 151 proteins were downregulated in trm5 ( Figure 5A). GO annotation of these differentially accumulating proteins revealed enrichment of the GO terms thylakoid, chloroplast, and photosystem I ( Figure 5B). KEGG annotation revealed enrichment of proteins involved in photosynthesis, and photosynthetic proteins ( Figure   5C). Taken together, the GO and KEGG analysis demonstrated that most differentially accumulating proteins are involved in processes related to photosynthesis.
Defects in tRNA m 1 G methylation can be expected to affect mRNA translation, particularly of proteins that have high numbers of affected codons. Therefore, we were interested in identifying genes that showed reduced expression at the protein level in our proteomics analysis of trm5 plants, but no detectable reduction in mRNA abundance. To identify such mRNAs, we performed RNA-seq on wild type and trm5 plants. We identified 1186 transcripts that were reduced in abundance in trm5 and 580 transcripts that were increased in abundance by at least 2 fold and hierarchically clustered these transcripts (Supplementary Figure S4 and Supplementary Table S4).
Comparison of the RNA-seq and proteomics datasets identified 133 proteins with reduced abundance, but with no detectable reduction in mRNA abundance (Supplementary Figure S4). We further inspected the data by selecting four candidate proteins with the highest fold change reported in the proteomics data (Table 1). From the selected candidate proteins, we discovered that three of the differentially expressed proteins reported in the proteomics data were not differentially expressed in the RNA-seq data, indicating that there is no change in transcripts levels leading to the fluctuation of their corresponding proteins. Only one protein candidate (AT2G45180.1) showed decreased fold change of mRNA, which correlates with the decrease of its corresponding protein reported in the proteomics data.  Figure S4).

AtTRM5 is involved in leaf and root development and flowering time regulation
Before undertaking growth measurements, we grew wild-type Columbia, trm5-1, two complemented lines, and two overexpression lines together under long-day conditions, harvested and dried the seeds to minimise any maternal or environmental effects. To observe the early growth stages of seedlings, we grew the six lines (wild type, trm5-1, two complemented lines and two overexpression lines) on plates for 10 days ( Figure 6A). The trm5-1 seedlings were noticeably smaller than the wild type. In contrast, no clear differences were evident between wild type, the complemented and overexpression lines. To rule out the possibility that the reduced growth in trm5-1 seedlings was due to slower germination, we measured the germination of trm5-1 and wild type and no difference was observed (Supplementary Figure S2). Reduced growth of trm5-1 roots was also evident on plate-grown plants ( Figure 6B).
Interestingly, trm5-1 primary, lateral and total (primary + lateral) root lengths were reduced in trm5-1 when compared to wild-type plants (Supplementary Figure S3). We also measured the lateral root number and found that trm5-1 plants had reduced numbers when compared to the wild type (Supplementary Figure S3). In contrast, no differences in the root growth were evident upon comparison of the wild type and the complemented lines. In TRM5 overexpression lines, primary and lateral root lengths were slightly longer than in the wild type ( Figure 6B and Supplementary Figure S3 Figure 7B). Together these results support a role for TRM5 in plant growth, development, and flowering time regulation.

Discussion
The discovery of m 1 G and m 1 I at position 37 in tRNAs of a wide range of eukaryotic and prokaryotic organisms underscores its importance as a key regulator of tRNA function and, presumably, translation (11,20,23). Previous studies in bacteria have shown that m 1 G37 is required for translational fidelity (20,57,58) and that mutations in the enzymes catalyzing m 1 G37 severely impact growth or cause lethal phenotypes (11,23,29). In eukaryotes, m 1 G37 modification requires the methyltransferase TRM5.
Here we report that in plants, trm5 mutants have a 60% reduction in m 1 G and m 1 I levels, display severely reduced growth and delayed transition to flowering. This is somewhat similar to human patients that are heterozygous for one mutant and one functional allele of Trm5. They show childhood failure to thrive and exercise intolerance symptoms (11).
With the 60% reduction of m 1 G in trm5 plants, it is reasonable to anticipate a significant We localized Arabidopsis TRM5 to the nucleus which is in contrast to the cytoplasmic and mitochondrial localisation in other eukaryotes: Trypanosoma brucei, Homo sapiens (HeLa cells) and Saccharomyces cerevisiae (11,23,29), although in one study S. cerevisiae Trm5 has also been localized exclusively to the nucleus (53).
Interestingly, in yeast, Trm5 acts in the nucleus to form m 1 G on retrograde imported tRNA Phe after initial export from the nucleus and subsequent splicing at the mitochondrial outer membrane. As retrograde tRNA import is conserved from yeast to vertebrates (59,60), it is tempting to speculate that TRM5-mediated m 1 G formation occurs in retrograde imported tRNAs in Arabidopsis.
Direct comparison of RNA-seq and proteomics data has been challenging as variation in protein abundance in proteomics datasets can be confounded by multiple factors.
Several factors can cause fluctuation in protein levels with no change of mRNA abundance: mRNA transcript abundance, translation rate, and translation resources availability (as reviewed in (as reviewed in (61)). This may explain the results of the initial comparison between our RNA-seq and proteomics data, and the seeming discrepancies in the derived sets of differentially expressed genes (Supplementary Figure S4). In our RNA-seq data, microtubule-related genes appeared to be significantly downregulated (AT1G21810, AT1G52410, AT2G28620, AT2G44190, AT3G23670, AT3G60840, AT3G63670, AT5G67270, AT4G14150, AT5G27000).
However, we could not detect any differential expression of these proteins in our proteomics dataset. This is not unexpected, as it has been reported that protein and transcript abundances only rarely correlate in data analysis (61). However, it has been shown that transcript abundance can still be used to infer protein abundance (62).
Among the selected four candidate proteins (Table 1) superfamily protein (AT1G03540.1) is unknown, but the protein was suggested to play a significant role in post-transcriptional modification of tRNAs (64). The P-glycoprotein 6 (AT2G39480) belongs to the large P-glycoprotein family and is known to play a role in mediating auxin transport, and disturbed auxin transport is known to affect plant growth (65). Our proteomics data suggested that the auxin transport is affected (AT5G35735, AT3G07390, AT4G12980, AT5G35735) in the trm5 mutant. Ferric reduction oxidase 8 (AT5G50160/AtFRO8) has been shown to participate in iron reduction and was implicated in leaf vein transport (66). We proposed that disturbed photosynthesis in trm5 mutant plants is a secondary effect of dysregulated transport processes.

20
The role of post-transcriptional RNA modifications in tRNA and mRNA metabolism and their impact on plant growth and development in plants are only beginning to be elucidated. Here, we have described the TRM5-mediated m 1 G methylation in tRNAs and identified crucial links between this modification, photosynthesis, and plant growth. It appears likely that the many other tRNA modifications in plant tRNAs also play important roles in translation and/or translational regulation which remain to be discovered.

Data Access
The data sets supporting the results of this article are available in NCBI's GEO database repository and are accessible through GEO Series accession number GSE114898. Proteomics raw data has been deposited on iProX, with under accession number IPX0001222000.   Position 37 in the gDNA is an adenine. In wild-type cDNA, a thymine was detected, in tad1-2 cDNA an adenine was observed and in trm5-1 a guanine was detected.