Rapid Genetic Code Evolution in Green Algal Mitochondrial Genomes

Abstract

Genetic code deviations involving stop codons have been previously reported in mitochondrial genomes of several green plants (Viridiplantae), most notably chlorophyte algae (Chlorophyta). However, as changes in codon recognition from one amino acid to another are more difficult to infer, such changes might have gone unnoticed in particular lineages with high evolutionary rates that are otherwise prone to codon reassignments. To gain further insight into the evolution of the mitochondrial genetic code in green plants, we have conducted an in-depth study across mtDNAs from 51 green plants (32 chlorophytes and 19 streptophytes). Besides confirming known stop-to-sense reassignments, our study documents the first cases of sense-to-sense codon reassignments in Chlorophyta mtDNAs. In several Sphaeropleales, we report the decoding of AGG codons (normally arginine) as alanine, by tRNA(CCU) of various origins that carry the recognition signature for alanine tRNA synthetase. In Chromochloris, we identify tRNA variants decoding AGG as methionine and the synonymous codon CGG as leucine. Finally, we find strong evidence supporting the decoding of AUA codons (normally isoleucine) as methionine in Pycnococcus. Our results rely on a recently developed conceptual framework (CoreTracker) that predicts codon reassignments based on the disparity between DNA sequence (codons) and the derived protein sequence. These predictions are then validated by an evaluation of tRNA phylogeny, to identify the evolution of new tRNAs via gene duplication and loss, and structural modifications that lead to the assignment of new tRNA identities and a change in the genetic code.

Keywords: Chlorophyta; Sphaeropleales; codon reassignment; genetic code; mitochondria; tRNA evolution.

Fig. 1.

Sense-to-sense codon reassignments predicted by CoreTracker in Viridiplantae. The species tree on the left is obtained using the combined information from multiple sources (see Materials and Methods). Numbers in rectangles indicate, for each mitochondrial genome, the use of the corresponding codon in protein-coding genes, ignoring intronic regions. Empty gray rectangles indicate that the codon is entirely avoided in the corresponding genome and colored rectangles indicate the decoding of the codon.

Fig. 2.

The consensus secondary structure of chlorophyte mitochondrion-encoded Ala-tRNA(UGC) is shown on the left. tRNA identity determinants for alanine are indicated by purple boxes and circle. On the right side, it is shown that the mtDNA-encoded tRNA(CCU) in Sphaeropleales and tRNA(CUA) in Neochloris aquatica and Hydrodictyaceae display multiple Ala-tRNA identity determinants, namely the G3:U70 invariant base pair, part of the usually conserved G¹GGC⁴ sequence, and A73 at the discriminator position. These results are consistent with the predicted AGG(Arg → Ala) in these Sphaeropleales and with UAG(Stop → Ala) in N. aquatica and Hydrodictyaceae. All tRNAs(CCU) are also missing the extra arm that characterizes Ser-tRNA (Bilokapić et al. 2009; Tukalo et al. 2013), confirming their Ala-tRNA identity.

Fig. 3.

Unrooted phylogenetic network of 321 tRNAs from seven groups (Met, Arg, Tyr, Cys, Ala, Leu, and Trp) constructed using the neighbor-net method as implemented in SplitsTree v4.14.6 (see Materials and Methods). Each group of tRNAs is shown with a different color and sequences that do not cluster with their group are displayed using the group color. For clarity, the label of some leaves that cluster with their respective tRNA isoacceptors is not shown. Mutant tRNAs that could potentially be involved in codon reassignments are shown in white, with a black background. Note that the identity of tRNAs under the standard decoding is preserved here to highlight their unusual affiliation.

Fig. 4.

Phylogeny of 30 core chlorophytes inferred using a supermatrix built from the properly translated sequences of the 13 standard mitochondrial proteins. The tree presented here is the majority-rule posterior consensus tree inferred with PhyloBayes under the CATGTR + GAMMA model. Both posterior probability (pp) from the Bayesian analyses (left value) and bootstrap support from RAxML LG + GAMMA maximum likelihood reconstruction (right value) are shown. A dash (–) is used to indicate when a branch is not present in the ML tree. Internal nodes without any label have a parental branch with a PP value of 1.0 and a bootstrap support >95%. Branch lengths are shown and correspond to the estimated number of amino acid substitutions per site. The tree is rooted using Mesostigma and Chlorokybus as outgroups. Known deviations of the mitochondrial code are also indicated, with the standard genetic code assumed to be used at the root of the phylogeny.

Fig. 5.

Evolutionary history of tRNA(YCU) in green plants. The usage of AGR codons is shown in the first two columns, with a dash used to indicate when the codon is missing in the coding regions of the considered genome. The presence/absence of tRNA(YCU) in each genome is also shown (third column). Moreover, the gain and loss history of tRNA(YCU) in each lineage during their mitochondrial evolution is indicated on the phylogeny.