A plastid without a genome: evidence from the nonphotosynthetic green algal genus Polytomella

Abstract

Polytomella spp. are free-living, nonphotosynthetic green algae closely related to the model organism Chlamydomonas reinhardtii. Although colorless, Polytomella spp. have a plastid, but it is still unknown whether they harbor a plastid genome. We took a next generation sequencing approach, along with transcriptome sequencing, to search for a plastid genome and an associated gene expression system in Polytomella spp. Illumina sequencing of total DNA from four Polytomella spp. did not produce any recognizable plastid-derived reads but did generate a large number of mitochondrial DNA sequences. Transcriptomic analysis of Polytomella parva uncovered hundreds of putative nuclear-encoded, plastid-targeted proteins, which support the presence of plastid-based metabolic functions, similar to those observed in the plastids of other nonphotosynthetic algae. Conspicuously absent, however, were any plastid-targeted proteins involved in the expression, replication, or repair of plastid DNA. Based on these findings and earlier findings, we argue that the Polytomella genus represents the first well-supported example, to our knowledge, of a primary plastid-bearing lineage without a plastid genome.

Figure 1.

Tree of chlorophycean and trebouxiophycean green algae with examples of species that have lost photosynthetic capabilities. Photosynthetic species are shown in green, and nonphotosynthetic species are shown in red. The cartoon beside the species name shows basic cellular structure (not to scale). Branching order is based on published phylogenetic analyses (Nakada et al., 2008; Smith et al., 2013b).

Figure 2.

Number of nuclear genes encoding potential plastid-targeted proteins in different functional categories determined from the P. parva transcriptome. The P. parva transcriptome is available from the Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis database (http://camera.calit2.net/) under project ID MMETSP0052_2. Annotations were performed by the National Center for Genome Resources following the standard protocol of the Marine Microbial Transcriptome Initiative (http://marinemicroeukaryotes.org/). Annotated transcripts were scanned for plastid-like functions. The C. reinhardtii plastid proteome, which contains 996 experimentally chloroplast-localized proteins (Terashima et al., 2011), and all presumed plastid-targeted proteins from the C. reinhardtii nuclear genome assembly (version 5.3.1; downloaded from Phytozome version 9.1) were also used to search (with BLASTp) the P. parva transcriptome for putative plastid proteins. All hits to the P. parva transcriptome were collected. Duplicates and hits corresponding to well-known nuclear or mitochondrial proteins were removed; however, some nuclear or mitochondrial proteins still remain in the list, because they may represent proteins with dual functions (Terashima et al., 2011).

Figure 3.

Venn diagram of plastid DNA-encoded genes in photosynthetic chlamydomonadalean algae and the nonphotosynthetic trebouxiophycean Helicosporidium sp. The chlamydomonadalean plastid genome (plastome) gene content (black line) is based on the following photosynthetic species: C. reinhardtii, Gonium pectorale, Pleodorina starrii, Volvox carteri, and Dunalielia salina (Fig. 1). A dashed line encloses the Helicosporidium sp. ptDNA genes. Genes numbered with black circles: (1) genes are nuclear encoded in C. reinhardtii and Polytomella spp.; (2) ycf1 in Arabidopsis (Arabidopsis thaliana) is known to encode a crucial component of the TIC complex required for plastid protein import (Kikuchi et al., 2013), and ycf2 is predicted to encode a subunit of protease enzyme involved in plastid protein degradation (Sakamoto 2006; Wagner et al., 2012) but may have a role in plastid gene expression, protein import, and/or the linkage of ptDNA to proteins or membranes (Bock, 2007; Wicke et al., 2011); and (3) like ycf2, data suggest that clpP encodes a subunit of a protease, which might be involved in the degradation of the chloroplast cytochrome b6f complex (Majeran et al., 2000) but may not be crucial for nonphotosynthetic plants and algae (Cahoon et al., 2003; de Koning and Keeling, 2006).