Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

Abstract

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.

Figure 1.

Degeneracy of the genetic code in plastids. Shown are the 16 codon boxes and the amino acids specified by the codons in each box. White codon boxes are served by a single tRNA species, light gray boxes by two tRNAs, dark gray boxes by three tRNAs and the black box by four tRNAs. The tRNA species are indicated by the triplet their anticodon has a perfect match with.

Figure 2.

Targeted deletion of the plastid trnfM gene. (A) Physical map of the trnfM-containing region in the tobacco plastid genome (ptDNA; 56). Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. (B) Map of the transformed plastid genome (transplastome) produced with plastid transformation vector pΔtrnfM. The selectable marker gene aadA (spotted box; 23) is driven by the plastid psbA promoter (PpsbA) and the 3′-UTR from the plastid rbcL gene (TrbcL; gray boxes). (C) RFLP analysis of ΔtrnfM chloroplast transformants. The transplastomic lines remain heteroplasmic and show both the 1.5 kb wild type-specific hybridization band and the 2.9 kb band diagnostic of the transplastome. Wt: wild type. (D) Phenotype of a typical (heteroplasmic) ΔtrnfM transplastomic plant. Arrows point to examples of misshapen leaves. (E) Example of a seed assay confirming heteroplasmy of ΔtrnfM plants and gradual loss of the transplastome in the absence of antibiotic selection. The transplastome is lost from most seedlings as evidenced by their white phenotype upon germination on spectinomycin-containing medium. The arrow points to one of the occasionally appearing green (spectinomycin-resistant) seedlings that still harbor the transplastome.

Figure 3.

Targeted inactivation of the plastid trnM gene. (A) Physical map of the trnM-containing region in the tobacco ptDNA. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites relevant for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transplastome produced with plastid transformation vector pΔtrnM-s. The selectable marker gene aadA is inserted into trnM gene in the same transcriptional orientation. The aadA coding region is shown as spotted box, the expression elements driving it as gray boxes. (C) Map of the transplastome produced with plastid transformation vector pΔtrnM-as. The selectable marker gene aadA is inserted into trnM gene in the opposite (antisense) orientation. (D) RFLP analysis of ΔtrnM plastid transformants. Both sets of transplastomic lines produced (ΔtrnM-s and ΔtrnM-as) remain heteroplasmic as evidenced by simultaneous presence of the 2.1 kb wild type-specific hybridization band and the 3.6 kb band diagnostic of the transplastome. Wt: wild type. (E) Phenotype of a typical ΔtrnM transplastomic plant. Arrows point to typical misshapen leaves. (F) Segregation analysis of a ΔtrnM plant. Seeds from a selfed transplastomic plant were sown on spectinomycin-containing medium. Spectinomycin sensitivity of most seedlings suggests a strong tendency to lose the transplastome in the absence of antibiotic selection. Examples of spectinomycin-resistant seedlings (that have retained the transplastome) are indicated by arrows.

Figure 4.

Disruption of the plastid trnI-CAU gene. (A) Physical map of the region in the tobacco plastid genome containing trnI-CAU. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The bent arrows indicate the borders of the transformation plasmid. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome (transplastome) produced with plastid transformation vector pΔtrnI-CAU. The aadA coding region is shown as spotted box, the expression elements driving it as gray boxes. (C) RFLP analysis of ΔtrnI-CAU transplastomic plants. All lines remain heteroplasmic as evidenced by a constant ratio of the 1.9 kb wild type-specific hybridizing fragment and the 3.5 kb fragment diagnostic of the transplastome. Wt: wild type. (D) Phenotype of a typical ΔtrnI-CAU transplastomic plant. Arrows point to misshapen leaves. (E) Inheritance assay of a ΔtrnI-CAU plant. Examples of spectinomycin-resistant seedlings that have retained the transplastome are indicated by arrows.

Figure 5.

Targeted disruption of the plastid trnI-GAU gene. (A) Physical map of the region in the tobacco plastome containing trnI-GAU. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. An origin of replication located in this region is denoted as oriA. (B) Map of the transformed plastid genome produced with plastid transformation vector pΔtrnI-GAU. The aadA coding region is shown as spotted box, the expression elements driving it as gray boxes. (C) RFLP analysis of ΔtrnI-GAU plastid transformants. The transplastomic lines remain heteroplasmic and show both the 4.4 kb wild type-specific hybridization band and the 5.9 kb band diagnostic of the transplastome. Wt: wild type. (D) Phenotype of a typical heteroplasmic ΔtrnI-GAU plant. Misshapen leaves are indicated by arrows. (E) Example of a seed assay confirming heteroplasmy of ΔtrnI-GAU plants and demonstrating loss of the transplastome in the absence of antibiotic selection. Arrows points to two green (spectinomycin-resistant) seedlings that still harbor the transplastome.

Figure 6.

Characteristic sequence features of the analyzed plastid tRNAs. (A) Secondary structures of the Nicotiana tabacum tRNA^Met(CAU), tRNA^fMet(CAU), tRNA^Ile(CAU) and tRNA^Ile(GAU). The tRNAs were folded using the ARAGORN webservice (57). The − indicates canonical base pairing, + denotes a GU base pair. The anticodons and the nucleotides in tRNA^Met(CAU) and tRNA^fMet(CAU), which are conserved (according to 42) are shown in bold. Additional characteristic sequence features are boxed and numbered (see text for details). The lysidine modification in the anticodon of tRNA^Ile(CAU) is indicated by an L. (B) List of distinguishing features that allow discrimination between the four tRNA species. Features are numbered as in (A). Features that were described as discriminatory in bacteria, but may not be so in plastids are in parentheses (see text for details). Cases in which the anticodon is probably the main discriminatory feature are also indicated.

Figure 7.

Codon recognition in the AUN codon box in bacteria, plastids and metazoan mitochondria. tRNA species are denoted by the first nucleotide of their anticodon (which pairs with the third codon position, N₃). Isoleucine-specifying triplets are shown as white boxes, methionine-specifying triplets as gray boxes. Note that AUA specifies Ile in bacteria and plastids, but Met in metazoan mitochondria. Naturally existing tRNA species are indicated for bacteria, plastids and mitochondria. Hypothetical tRNA species whose existence would lead to decoding ambiguities (due to forbidden wobble and superwobble interactions) are shown in parentheses. C* = lysidine, C** = 5-formylcytidine.