Euglenophytes are a familiar algal group with green alga-derived secondary plastids, but the knowledge of euglenophyte plastid function and evolution is still highly incomplete. With this in mind we sequenced and analysed the transcriptome of the non-photosynthetic species Euglena longa. The transcriptomic data confirmed the absence of genes for the photosynthetic machinery, but provided candidate plastid-localised proteins bearing N-terminal bipartite topogenic signals (BTSs) of the characteristic euglenophyte type. Further comparative analyses including transcriptome assemblies available for photosynthetic euglenophytes enabled us to unveil salient aspects of the basic euglenophyte plastid infrastructure, such as plastidial targeting of several proteins as C-terminal translational fusions with other BTS-bearing proteins or replacement of the conventional eubacteria-derived plastidial ribosomal protein L24 by homologs of archaeo-eukaryotic origin. Strikingly, no homologs of any key component of the TOC/TIC system and the plastid division apparatus are discernible in euglenophytes, and the machinery for intraplastidial protein targeting has been simplified by the loss of the cpSRP/cpFtsY system and the SEC2 translocon. Lastly, euglenophytes proved to encode a plastid-targeted homolog of the termination factor Rho horizontally acquired from a Lambdaproteobacteria-related donor. Our study thus further documents a substantial remodelling of the euglenophyte plastid compared to its green algal progenitor.

The chloroplasts of Euglena gracilis bounded by three membranes arose via secondary endosymbiosis of a green alga in a heterotrophic euglenozoan host. Many genes were transferred from symbiont to the host nucleus. A subset of Euglena nuclear genes of predominately symbiont, but also host, or other origin have obtained complex presequences required for chloroplast targeting. This study has revealed the presence of short introns (41–93 bp) either in the second half of presequence-encoding regions or shortly downstream of them in nine nucleus-encoded E. gracilis genes for chloroplast proteins (Eno29, GapA, PetA, PetF, PetJ, PsaF, PsbM, PsbO, and PsbW). In addition, the E. gracilis Pbgd gene contains two introns in the second half of presequence-encoding region and one at the border of presequence-mature peptide-encoding region. Ten of 12 introns present within presequence-encoding regions or shortly downstream of them identified in this study have typical eukaryotic GT/AG borders, are T-rich, 45–50 bp long, and pairwise sequence identities range from 27 to 61%. Thus single recombination events might have been mediated via these cis-spliced introns. A double crossing over between these cis-spliced introns and trans-spliced introns present in 5′-UTRs of Euglena nuclear genes is also likely to have occurred. Thus introns and exon-shuffling could have had an important role in the acquisition of chloroplast targeting signals in E. gracilis. The results are consistent with a late origin of photosynthetic euglenids.