The complete plastome of the coccoid green alga Jenufa minuta (Chlorophyceae, incertae sedis) unveils a noncanonical genetic code and a previously unrecognized trans-spliced group II intron in the rpl32 gene

Abstract

We describe the plastome of Jenufa minuta, a unicellular green alga whose precise relationship with the Sphaeropleales (Chlorophyceae) remains uncertain. This 206,680-bp genome contains 100 conserved genes, 11 of which exhibit 16 group I and seven group II introns. We discovered a trans-spliced group II intron in the rpl32 gene of J. minuta and its orthologs in all Sphaeropleales and several Chlamydomonadales; this is the first time this intron is documented. As in the mitochondria of J. minuta, the UGA codon is decoded as tryptophan. To our knowledge, this is the second report of a variant genetic code in plastids.

Keywords:

• Noncanonical genetic code
• Sphaeropleales
• trans-spliced group II intron
• UGA codon reassignment

Departures from the standard genetic code arose more frequently in mitochondria than in plastids during the evolution of photosynthetic algae. Among green algae, various noncanonical genetic codes have been observed in mitochondria, particularly in compact mitogenomes that underwent reduction in gene content; e.g. in those of the prasinophyte Pycnococcus provasolii (Turmel et al. 2010), representatives of the Pedinomonadales and Marsupiomonadales (Pedinophyceae) (Turmel et al. 1999; Turmel, Otis, Lemieux 2020), and several members of the Sphaeropleales (Chlorophyceae) (Noutahi et al. 2019; Zihala and Elias 2019). In contrast, evidence for a variant genetic code (i.e. reassignment of AUA from isoleucine to methionine) in the plastome has been reported only for the tiny prasinophytes belonging to the genus Chloroparvula (Chloropicophyceae) (Turmel et al. 2019). Here, we present the plastome sequence of Jenufa minuta (Chlorophyceae, incertae sedis) and provide evidence for a second case of noncanonical genetic code. This chlorophycean alga, whose mitogenome also exhibits a variant genetic code (Turmel, Otis, Vincent, et al. 2020), belongs to a lineage that is sister to Sphaeropleales or represents a deep lineage of the latter order (Lemieux et al. 2015).

J. minuta (CAUP H8102) was obtained from the Culture Collection of Algae at Charles University in Prague. Total cellular DNA was sequenced on the Roche 454 platform by the Genomic Analysis Platform of Laval University. The methods of DNA extraction, sequencing and assembly of the plastome sequence have been described previously (Lemieux et al. 2015).

The 206,680-bp plastome of J. minuta (GenBank NC_028582) displays a 11,100-bp inverted repeat that contains only the rDNA operon. The repertoire of 100 different conserved genes comprises 29 tRNA and 68 protein-coding genes (including rpl12, a gene present in Sphaeropleales but missing in other chlorophycean orders). Coding regions of 11 genes are interrupted by 16 cis-spliced group I introns and seven group II introns, three of which are trans-spliced. Two of the latter introns occur in psaA and the other in rpl32 only 13 nucleotides downstream of the initiator codon. This is the first time that a trans-spliced intron is reported in rpl32 among green algae. Upon reanalysis of complete chlorophycean plastomes, we identified this trans-spliced intron in all Sphaeropleales and several Chlamydomonadales. Modeling of the J. minuta intron secondary structure revealed that the breakpoint falls within domain IV.

The UGA codon is a sense codon in the J. minuta plastid. It resides at 64 positions within the coding regions of 12 protein-coding genes and is decoded as tryptophan according to the alignment of the protein products against their green algal homologs. UGA is also decoded as tryptophan in the mitochondria of J. minuta and Jenufa perforata (Turmel, Otis, Vincent, et al. 2020). The trnW(uca) gene was not detected in the J. minuta plastome; hence, it is unclear which tRNA recognizes the UGA codon in the plastid.

Phylogenetic analysis of 55 plastome-encoded proteins from 33 chlorophycean algae using RAxML v.8.2.3 (Stamatakis 2014) recovered a strongly supported clade containing J. minuta and J. perforata, which is sister to most Sphaeropleales (Figure 1).

Figure 1. RAxML tree inferred from 55 concatenated plastome-encoded proteins of 33 chlorophycean green algae. Oltmannsiellopsis viridis and Pseudendoclonium akinetum (Ulvophyceae) were used as outgroup taxa. The figure shows the best-scoring tree, with bootstrap support values (100 replicates) reported on the nodes. GenBank accession numbers are provided for the plastomes of all taxa. The scale bar denotes the estimated number of amino acid substitutions per site. The data set was generated using the predicted protein sequences derived from atpA, atpB, atpE, atpF, atpH, atpI, ccsA, cemA, clpP, petB, petD, petG, petL, psaA, psaB, psaC, psaJ, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ, rbcL, rpl2, rpl5, rpl14, rpl16, rpl20, rpl23, rpl36, rpoA, rpoB, rpoC2, rps3, rps4, rps7, rps8, rps9, rps11, rps12, rps14, rps18, rps19, tufA, ycf3. Following alignment of the sequences of individual proteins with Muscle v3.7 (Edgar 2004), ambiguously aligned regions were removed using TrimAL v1.4 (Capella-Gutierrez et al. 2009) with the options block = 6, gt = 0.7, st = 0.005 and sw = 3, and the protein alignments were concatenated using Phyutility v2.2.6 (Smith and Dunn 2008). The phylogenetic analysis was carried out under the GTR + Γ4 model.

Disclosure statement

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada under Grants RGPIN-2007-2830 and RGPIN-2017-04506.