Extensive import of nucleus-encoded tRNAs into chloroplasts of the photosynthetic lycophyte, Selaginella kraussiana

Abstract

Over the course of evolution, land plant mitochondrial genomes have lost many transfer RNA (tRNA) genes and the import of nucleus-encoded tRNAs is essential for mitochondrial protein synthesis. By contrast, plastidial genomes of photosynthetic land plants generally possess a complete set of tRNA genes and the existence of plastidial tRNA import remains a long-standing question. The early vascular plants of the Selaginella genus show an extensive loss of plastidial tRNA genes while retaining photosynthetic capacity, and represent an ideal model for answering this question. Using purification, northern blot hybridization, and high-throughput tRNA sequencing, a global analysis of total and plastidial tRNA populations was undertaken in Selaginella kraussiana. We confirmed the expression of all plastidial tRNA genes and, conversely, observed that nucleus-encoded tRNAs corresponding to these plastidial tRNAs were generally excluded from the chloroplasts. We then demonstrated a selective and differential plastidial import of around forty nucleus-encoded tRNA species, likely compensating for the insufficient coding capacity of plastidial-encoded tRNAs. In-depth analysis revealed differential import of tRNA isodecoders, leading to the identification of specific situations. This includes the expression and import of nucleus-encoded tRNAs expressed from plastidial or bacterial-like genes inserted into the nuclear genome. Overall, our results confirm the existence of molecular processes that enable tRNAs to be selectively imported not only into mitochondria, as previously described, but also into chloroplasts, when necessary.

Keywords: RNA trafficking; evolution; gene transfer; oorganelle; plant.

Fig. 1.

S. kraussiana plastids contain plastid-encoded and nucleus-encoded tRNAs. (A) Summary of plastidial tRNA gene content in the model angiosperm, A. thaliana, and in three lycophyte species representative of the orders Lycopodiales (Huperzia Serrata), Isoetales (Isoetes flaccida), and Selaginellales (S. kraussiana). Filled squares indicate the presence of an intact tRNA gene sequence with gene names based on the one-letter amino acid code in uppercase and the anticodon in lowercase. Elongator and initiator methionine tRNAs are abbreviated Mcat and iMcat respectively. (B) Design of the experiments performed here. (C) Hybridization of probes specific to plastid-encoded p5S rRNA, ptRNA^Phe, and ptRNA^Tyr and to nucleus-encoded ntRNA^Phe, ntRNA^Thr(CGT), and ntRNA^Ala(AGC) to northern blots of total (T) or plastidial (P) RNAs. For each tRNA, the observed localization is indicated to the right.

Fig. 2.

Widespread and differential import of nucleus-encoded tRNAs into plastids of S. kraussiana. The tRNAs from plastid and nucleus fractions were sequenced and analyzed with mim-tRNAseq, and log₂ Fold Change (Plastid/Total) was obtained with DESeq2. Only tRNAs with a base mean value >100, a mean number of reads in total fractions >10, and Padj<0.05 are shown. (A) Log₂ Fold Change (Plastid/Total) of tRNA isodecoders. Green indicates plastid-encoded tRNAs, and black shows their nucleus-encoded counterparts. Blue hatches represent the minimum set of tRNAs required to complement the incomplete set of tRNAs of plastidial origin (for each codon, the most enriched nucleus-encoded tRNA isodecoder was selected); the remaining tRNAs are indicated in blue. tRNA isodecoder numbers are attributed by the mim-tRNAseq program. (B) Percentage of plastid-encoded tRNAs (green) versus their nucleus-encoded counterparts (black) in the plastidial (P) fractions (n = 3). tRNAs are named based on their cognate amino acids. For comparison, the percentage of plastid-encoded tRNA^Tyr (green) versus its nucleus-encoded counterpart (black) in the total (T) fractions is indicated (n = 3). Percentages were calculated from the mean of normalized mim-tRNAseq read counts per million of reads in the three plastidial or the three total libraries. (C) Universal genetic code showing the decoding capacity of the plastid-encoded (green box) and the imported nucleus-encoded tRNAs (blue box). Light green and orange boxes indicate the coding capacity of plastid-like (plike) and bacterial-like (blike) nucleus-encoded tRNAs respectively. (D) Log₂ Fold Change (Plastid/Total) of nucleus-encoded tRNA isodecoder families. For each isodecoder family, the blue dot shows the most enriched tRNA, the other tRNAs of the family being depicted with gray dots. The cases of tRNA^Pro, tRNA^Arg, and tRNA^Ile discussed in the manuscript are highlighted with green, gray, and blue frames respectively. (E) For each tRNA family presented in D, the Log₂ Fold Change (Plastid/Total) difference between the most and least enriched tRNAs (y-axis) is presented as a function of the number of nucleotide differences (x-axis). Plike tRNA^Pro and blike tRNA^Arg are highlighted with red triangles.

Fig. 3.

Nucleus-encoded tRNAs imported into plastids have different genetic origins. (A) On the Left, a phylogenetic tree shows two evolutionarily distant tRNA^Pro gene groups that coexist in S. kraussiana. Each group contains genes coding for tRNA^Pro isoacceptors with AGG, CGG, and UGG anticodons. One group (blue arrows) is of eukaryotic origin, the other group (green arrows) has a plastidial origin. In the middle, a phylogenetic tree showing the bacterial origin of a single tRNA^ArgUCU gene (black arrow). The other tRNA^ArgUCU genes have eukaryotic origins (see also SI Appendix, Fig. S7). On the Right, a phylogenetic tree demonstrates that nucleus-encoded tRNA^IleCAU genes (blue arrows) are derived from the tRNA^IleAAU gene of eukaryotic origin. The tRNA^IleCAU-3/4 isodecoders, which are nearly identical to tRNA^IleCAU-2, are not represented on the tree. Nomenclature of plant species is as described in (4). Bacteria nomenclature is as follows: Cac, Candidatus atelocyanobacterium; Cya, Cyanobacterium stanieri; Mor, Moraxella catharrhalis; Tur, Turicibacter sanguinis; Ric, Rickettsia prowezakii; Apr, Alpha proteobacterium HIMB5; Syn, Syntrichia caninervis. Extended phylogenetic trees are presented in SI Appendix, Fig. S8. (B) On the Left, histograms show the normalized mim-tRNAseq read counts per million of reads (RPM) of tRNA^Pro AGG, CGG, and UGG, in the total (T) or plastidial (P) fractions. Error bars represent the SD of read counts per million in the six libraries. tRNA^Pro isoacceptors of eukaryotic origin have been divided into tRNA isodecoder families (named based on their anticodon and their numbering in the mim-tRNAseq). Blue bars represent tRNAs of eukaryotic origin, and green bars represent tRNAs of plastidial origin. In the middle and on the Right, histograms for tRNA^ArgUCU and tRNA^IleCAU and AAU are shown, as described for tRNA^Pro species.

Fig. 4.

An evolutionary model describing the different means used by the photosynthetic plant S. kraussiana to ensure functional protein synthesis in the cytosol and plastids. In the course of evolution, on one hand, new functional nuclear genes have been acquired by intracellular transfer (IGT) of a plastidial gene (tRNA^ProUGG) or by horizontal transfer (HGT) of a bacterial gene (tRNA^ArgUCU). In this latter case, acquisition by IGT of a mitochondrial gene cannot be ruled out and is also hypothesized. Duplication of nuclear tRNA genes, followed by mutations, particularly at the anticodon, has led to the generation of new tRNAs (e.g., tRNA^IleCAU). On the other hand, several plastidial tRNA genes have been lost and their lack is now compensated by the import of nucleus-encoded tRNAs of various origins (eukaryotic, plastidial, and bacterial or mitochondrial) into chloroplasts. This import is selective and not all nucleus-encoded tRNAs are found in the organelle.