Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors

Abstract

Chloroplasts originate through the endosymbiotic integration of a host and a photosynthetic symbiont, with processes established within the host for the biogenesis and maintenance of the nascent chloroplast. It is thought that several photosynthetic eukaryotes have replaced their original chloroplasts with others derived from different source organisms in a process termed "serial endosymbiosis of chloroplasts." However, it is not known whether replacement chloroplasts are affected by the biogenesis and maintenance pathways established to support their predecessors. Here, we investigate whether pathways established during a previous chloroplast symbiosis function in the replacement chloroplasts of the dinoflagellate alga Karenia mikimotoi. We show that chloroplast transcripts in K. mikimotoi are subject to 3' polyuridylylation and extensive sequence editing. We confirm that these processes do not occur in free-living relatives of the replacement chloroplast lineage, but are otherwise found only in the ancestral, red algal-derived chloroplasts of dinoflagellates and their closest relatives. This indicates that these unusual RNA-processing pathways have been retained from the original symbiont lineage and made use of by the replacement chloroplast. Our results constitute an addition to current theories of chloroplast evolution in which chloroplast biogenesis may be radically remodeled by pathways remaining from previous symbioses.

Fig. 1.

Oligo(dA) and gene-specific RT-PCRs for transcripts from K. mikimotoi, A. carterae, E. huxleyi, and P. tricornutum. The gel photographs display the products from a series of RT-PCRs to detect polyuridylylated transcripts from K. mikimotoi as well as representative haptophyte and diatom species. The size standard is DNA Hyperladder I (Bioline). Lanes 1–5: oligo(dA) RT-PCR of K. mikimotoi psbA, psbC, psbD, psaA, and rbcL. Lanes 6–7: oligo(dA) RT-PCR of K. mikimotoi PCNA and cox1. Lanes 8–10: gene-specific RT-PCR of K. mikimotoi psbA, PCNA, and cox1. Lanes 11–12: oligo(dA) RT-PCR of E. huxleyi psbA and psbD. Lanes 13–14: gene-specific RT-PCR of E. huxleyi psbA and psbD. Lanes 15–16: oligo(dA) RT-PCR of P. tricornutum psbA and psbD. Lanes 17–18: gene-specific RT-PCR of P. tricornutum psbA and psbD. Lane 19: oligo(dA) RT-PCR of A. carterae psbA. Lane 20: reverse transcriptase negative control for oligo(dA) RT-PCR of K. mikimotoi psbA.

Fig. 2.

Aligned 3′ ends of K. mikimotoi polyuridylylated chloroplast transcripts. This alignment shows the first 50 nt of the 3′ UTR of the K. mikimotoi psbA, psbC, psbD, psaA, and rbcL genes, labeled “gDNA.” The 3′ ends of sequenced, cloned polyuridylylated transcripts are shown below each gDNA sequence. Transcript sequences are shown for each unique poly(U) site observed. Only the first 18 nt of the poly(U) tract are shown for each transcript. Numbers in parentheses correspond to the full length of the poly(U) tract as sequenced in different clones. Specific sequences that are asterisked were obtained directly from crude RT-PCR products.

Fig. 3.

Base editing in K. mikimotoi chloroplast transcripts. (A) Tabulated base-editing events observed in K. mikimotoi psbA, psbC, psbD, psaA, and rbcL transcripts. Sequences were obtained using PCR forward primers against the 5′ end of each gene (Dataset S2). (B) The total extent of different types of base editing observed. (C) Poly(U)-associated base editing. The sequences cover positions 890–940 of K. mikimotoi psbA as obtained from (top to bottom): genomic DNA, cDNA generated from nonpolyuridylylated transcripts, and oligo(dA) cDNA. Although three editing events (at positions 897, 914, and 937, labeled with gray arrows) occur before transcript polyuridylylation, two events (at positions 909 and 918, labeled with black arrows) are specifically associated with polyuridylylated transcripts.