Transfer RNA modifications and genes for modifying enzymes in Arabidopsis thaliana - PubMed (original) (raw)

Transfer RNA modifications and genes for modifying enzymes in Arabidopsis thaliana

Peng Chen et al. BMC Plant Biol. 2010.

Abstract

Background: In all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant Arabidopsis thaliana, to understand the function of nucleoside modifications in plant growth and development.

Results: In this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, Arabidopsis thaliana and hybrid aspen (Populus tremula × tremuloides). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in Sacccharomyces cerevisiae and Escherichia coli, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family.Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m1G, m2G, m7G and ncm5U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm5U modification). The AtTRM11 mutant exhibited an early-flowering phenotype, and the AtELP1 mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots.

Conclusions: Using a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in Arabidopsis thaliana. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.

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Figures

Figure 1

Figure 1

Modified nucleosides in eukaryotic tRNAs and chemical structures. A: Clover-leaf structure of eukaryotic tRNA. Each circle represents a nucleotide, numbered from 5'- to 3'- end. Modified nucleosides found at different positions are shown. B: Chemical structures of some modified nucleosides.

Figure 2

Figure 2

HPLC chromatogram of modified nucleosides in tRNAs from Arabidopsis and Poplar. X-scale: retention time of modified nucleosides in minutes. Y-scale: UV absorbance at 254 nm. Peaks marked with black triangle represent plant-specific modified nucleosides.

Figure 3

Figure 3

Venn diagram showing similarities and differences of modified nucleosides between A. thaliana, S.cerevisiae and E. coli. Modified nucleosides were shown with abbreviations, comments (a)-(g) were the same as in Table 1.

Figure 4

Figure 4

Conserved domain of TRM11TAD1TAD2and TAD3 gene homologs in plants. Part of protein sequence alignments were shown with numbers above showing position from the first amino acid. A: Motif I within catalytic domain of TRM11 gene homologs, conserved residue D215 and D291 are marked with arrows. B: Deaminase domain of TAD1 gene homologs, conserved residue is marked with black arrow. C: Deaminase domain of TAD2 and TAD3 gene homologs, conserved residue is marked with black arrow.

Figure 5

Figure 5

T-DNA lines used in this study and corresponding genes. Gene models were shown with dark gray box representing exon and lines in between as intron. T-DNA insertion was shown as a triangle with NASC line name above. Relevant modified nucleosides for corresponding gene and HPLC results were indicated: "+" represents a change of amount of modified nucleoside in the mutant; "-" represents no change.

Figure 6

Figure 6

HPLC chromatogram of the T-DNA homozygous mutants defective in modified nucleosides. Parts of the HPLC chromatogram were shown with black triangle indicating position of the relevant modified nucleosides. NASC line number, allele number and modified nucleosides affected were shown in each panel, numbers above or within peaks represent retention time in minutes.

Figure 7

Figure 7

Phenotype of mutant plants. A: Early flowering of N622158 mutant plants and narrow leaf phenotype of N661341 mutant plants. Picture was taken at 21 D under LD condition. B and C: Serrated leaf shape of the third and fourth true leaves of N661341 mutant plant (C, indicated with arrows) compared to Col.0 (B). Picture was taken 13 D under LD condition. D and E: Reduced root growth of N661341 mutant plants (E) compared with Col.0 (D). MS plates were incubated vertically in tissue culture room for 8 D before picture was taken. F and G: N661341 mutant plant had problem of secondary shoot growth (G) compared to wild type (F). Plants were grown in LD conditions, primary shoots were cut at 3 weeks and picture taken at 6 weeks. H and I: N661341 mutant plant had aberrant silique shape (I) compared with Col.0 (H). Plants were grown in LD conditions.

Figure 8

Figure 8

Heat map of Arabidopsis tRNA modification candidate genes. A: All tRNA modification candidate genes in Arabidopsis found in this study. Data downloaded from AtGenExpress database where information for tissue cluster and sample ID can be found. The five tRNA modification genes identified in this study were marked with asterisk. B and C: Heat map of AtTRM82, AtKTI12, AtELP1, AtTRM10 and AtTRM11 from Developmental dataset (B) and Tiling dataset (C) from Tileviz database.

Figure 9

Figure 9

Phylogenetic tree of TRM10, TRM11, TRM82, ELP1 gene homologs in plants and KTI12 tree from all domains of life. Gene accession number and organism was shown, with branch numbers showing substitution rate per site for sequence alignment. A: Trm10 tree; B: Trm11 tree; C: Trm82 tree; D: Elp1 tree. E: Kti12 tree (representative of organism from all domains of life).

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