Organellar DNA Polymerases in Complex Plastid-Bearing Algae

Abstract

DNA replication in plastids and mitochondria is generally regulated by nucleus-encoded proteins. In plants and red algae, a nucleus-encoded enzyme called POP (plant and protist organellar DNA polymerase) is involved in DNA replication in both organelles by virtue of its dual localization. POPs are family A DNA polymerases, which include bacterial DNA polymerase I (PolI). POP homologs have been found in a wide range of eukaryotes, including plants, algae, and non-photosynthetic protists. However, the phylogeny and subcellular localizations of POPs remain unclear in many algae, especially in secondary and tertiary plastid-bearing groups. In this study, we report that chlorarachniophytes possess two evolutionarily distinct POPs, and fluorescent protein-tagging experiments demonstrate that they are targeted to the secondary plastids and mitochondria, respectively. The timing of DNA replication is different between the two organelles in the chlorarachniophyte Bigelowiella natans, and this seems to be correlated to the transcription of respective POP genes. Dinoflagellates also carry two distinct POP genes, possibly for their plastids and mitochondria, whereas haptophytes and ochrophytes have only one. Therefore, unlike plants, some algal groups are likely to have evolved multiple DNA polymerases for various organelles. This study provides a new insight into the evolution of organellar DNA replication in complex plastid-bearing organisms.

Keywords: DNA replication; algae; chlorarachniophytes; endosymbiosis; mitochondria; plastids.

Figure 1

Maximum likelihood phylogenetic tree of family A DNA polymerases, including 93 POPs (plant and protist organellar DNA polymerases). The tree was generated under the LG+R7 model implemented in IQ-TREE. The values at nodes indicate bootstrap supports calculated by the two different substitution models (LG+R7/LG+C20+F+Γ) when they are higher than 50%, and black dots on branches represent robust supports (>95%). The scale bars show the number of inferred amino acid substitutions per site. Accession numbers of GenBank, JGI (Joint Genome Institute)/, and MMETSP (Marine Microbial Eukaryotic Transcriptome Sequencing Project) are shown on the right side of species names.

Figure 2

Subcellular localization of BnPOP1 and BnPOP2. Green/yellow/cyan fluorescent protein (GFP/YFP/CFP) fusion proteins were expressed in the chlorarachniophyte, Amorphochlora amoebiformis. The red color is the chlorophyll-autofluorescence, and the yellow and green signal represent localization of BnPOP1 (A) and BnPOP2 (B), respectively. Mitochondria were labelled by CFP fused with the mitochondria-targeted histidine tRNA synthetase. The scale bars are 10 µm.

Figure 3

Timings of DNA replication for nuclear, mitochondrial, and plastid genomes. (A) Cell growth in temporally synchronized culture of Bigelowiella natans during a diurnal cycle. (B–D) Plots of changes in nuclear, mitochondrial, and plastid DNA levels, which were determined by real-time qPCR with two sets of specific primers (blue and red). (E,F) Relative transcript levels for BnPOP1 and BnPOP2. Error bars represent the standard deviation of triplicate qPCR reactions.