Functional Remodeling of RNA Processing in Replacement Chloroplasts by Pathways Retained from Their Predecessors

Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors Richard G. Dorrell1 and Christopher J. Howe Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom Edited* by J. Woodland Hastings, Harvard University, Cambridge, MA, and approved October 3, 2012 (received for review July 19, 2012) Chloroplasts originate through the endosymbiotic integration of stood that processes originating in the host play important roles a host and a photosynthetic symbiont, with processes established in chloroplast biogenesis and maintenance (2, 20, 21). This raises within the host for the biogenesis and maintenance of the nascent the question whether processes established during an earlier chlo- chloroplast. It is thought that several photosynthetic eukaryotes roplast symbiosis could be retained through serial endosymbiosis have replaced their original chloroplasts with others derived from and contribute to the function of the replacement chloroplast. different source organisms in a process termed “serial endosymbi- Furthermore, if such processes were made use of by replacement osis of chloroplasts.” However, it is not known whether replace- chloroplasts lacking them, the biology of the replacement might ment chloroplasts are affected by the biogenesis and maintenance be dramatically changed as a result. We therefore wanted to de- pathways established to support their predecessors. Here, we in- termine whether the biochemical activities of replacement chlor- vestigate whether pathways established during a previous chloro- oplasts could be functionally altered by pathways retained from plast symbiosis function in the replacement chloroplasts of the ancestral symbioses. dinoflagellate alga Karenia mikimotoi. We show that chloroplast We studied RNA processing in a model example of chloroplast transcripts in K. mikimotoi are subject to 3′ polyuridylylation and replacement, the dinoflagellate Karenia mikimotoi. This globally extensive sequence editing. We confirm that these processes do distributed species, implicated in the formation of fish-killing not occur in free-living relatives of the replacement chloroplast blooms (22), is a particularly well-studied example (6, 23, 24) of EVOLUTION lineage, but are otherwise found only in the ancestral, red algal- dinoflagellates containing the light-harvesting carotenoid pigment fl derived chloroplasts of dino agellates and their closest relatives. fucoxanthin. The majority of photosynthetic dinoflagellates harbor This indicates that these unusual RNA-processing pathways have red algal-derived chloroplasts, which contain the light-harvesting been retained from the original symbiont lineage and made use of carotenoid pigment peridinin and have an unusual, highly reduced by the replacement chloroplast. Our results constitute an addition genome, which is fragmented into a number of small plasmid-like to current theories of chloroplast evolution in which chloroplast elements termed “minicircles” (4, 11, 25–27). Fucoxanthin- biogenesis may be radically remodeled by pathways remaining containing dinoflagellates, by contrast, contain chloroplasts with a from previous symbioses. much less reduced genome that is not encoded on minicircles, although some subgenomic molecules have been reported (15, 28). organelle | fucoxanthin | haptophyte | poly(U) | alveolate The chloroplasts of fucoxanthin dinoflagellates group phylo- genetically with those of another group of algae, the haptophytes, hloroplasts originate through endosymbiosis, in which a free- to the exclusion of other lineages (7, 15, 16, 18). Although the Cliving photosynthetic symbiont is taken up by a eukaryotic phylogenetic relationship between the peridinin-containing host, with processes becoming established within the host to and fucoxanthin-containing chloroplast lineages has historically support the biogenesis and maintenance of the symbiont (1, 2). It proved controversial (6, 24), recent studies have confirmed that has long been understood that many such endosymbiotic events the peridinin-containing chloroplast lineage is the ancestral type have occurred across the eukaryotes, giving rise to a diverse array and was acquired before the radiation of photosynthetic dino- – of extant chloroplast lineages (2 4). Genomic and phylogenetic flagellates (15, 29). Thus, the fucoxanthin-containing chloroplasts evidence has suggested that several major chloroplast-containing were derived from a subsequent independent endosymbiotic event lineages have replaced their original chloroplasts with others that replaced the ancestral red-algal chloroplast in K. mikimotoi derived from a different evolutionary lineage in a process termed fl “ ” and related dino agellate lineages. serial endosymbiosis of chloroplasts (2, 5). The best-supported The ancestral chloroplasts of peridinin-containing dinoflagellates examples of chloroplast replacement are found within the di- fi fl show a number of unusual posttranscriptional RNA modi cations. no agellate algae, within which at least three distinct chloroplast These include the addition of 3′ terminal poly(U) tracts, and (in replacement events are known to have occurred (2, 6, 7). Two some species) extensive transcript editing (3, 29–33). Outside the even more dramatic hypotheses of chloroplast replacement have fl fi peridinin dino agellates, chloroplast RNA polyuridylylation has recently been put forward. The rst proposes that the ancestors so far been reported only in the closely related species Chromera of taxa currently harboring red algal-derived chloroplasts, such as diatoms and apicomplexan parasites, contained green algal symbionts (5, 8–11). In the second hypothesis, even the cya- Author contributions: R.G.D. and C.J.H. designed research; R.G.D. performed research; nobacterial-derived chloroplasts of plants and their closest rel- R.G.D. analyzed data; and R.G.D. and C.J.H. wrote the paper. atives are replacements for organelles derived from an The authors declare no conflict of interest. ancestral symbiosis involving a chlamydiobacterial symbiont (12, 13). Although both these proposed replacement events remain *This Direct Submission article had a prearranged editor. controversial (8, 14), serial endosymbioses may constitute Data deposition: Full-length DNA sequences, nonpolyuridylylated transcripts, and polyuridylylated transcripts for each of Karenia mikimotoi psbA, psbC, psbD, psaA,andrbcL a widespread feature of chloroplast evolution. genes have been deposited in the GenBank database (accession nos. JX899682– Regardless of the number of chloroplast replacement events JX899726). that have occurred, one outstanding question is whether, in any 1To whom correspondence should be addressed. E-mail: [email protected]. given instance, the ancestral chloroplast symbiosis might affect This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the biology of the incoming chloroplast (15–19). It is well under- 1073/pnas.1212270109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212270109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 velia (29), and chloroplast transcript editing has been reported species (Fig. 1). We found no evidence of polyuridylylated tran- only in land plants (3). Here, we report that these unusual scripts for the psbA or psbD genes of either species (Fig. 1, chloroplast transcript-processing pathways, derived from the an- lanes 11, 12, 15, 16), whereas positive control experiments using cestral peridinin-containing dinoflagellate chloroplast symbiosis, internal gene-specific primers yielded products of the expected function in the replacement chloroplasts of fucoxanthin-containing sizes (Fig. 1, lanes 13, 14, 17, 18). We confirmed the absence of dinoflagellates. This demonstrates that the biology of serial en- transcript polyuridylylation in each species through independent dosymbionts can be dramatically remodeled by host functions repeats of each oligo(dA) RT-PCR, using forward primers against remaining from previous symbioses. the E. huxleyi and P. tricornutum psbA, psbC, psbD, psaA,andrbcL genes (Fig. S1). Results We confirmed the results showing that transcripts of K. miki- 3′ Terminal Polyuridylylation of Chloroplast Transcripts in K. mikimotoi. motoi chloroplast genes were polyuridylylated by cloning and We first tested if transcripts in the serially acquired chloroplasts sequencing RT-PCR products for each reaction (Fig. 2). In each of K. mikimotoi carried 3′ poly(U) tracts. We generated cDNA clone sequenced, we observed poly(U) tracts similar to those pre- from total cellular RNA of K. mikimotoi using an oligo(dA) primer viously reported in peridinin dinoflagellates and C. velia (29, 31, and then carried out PCR reactions with oligo(dA) as a reverse 34). Each sequenced clone was polyuridylylated 8–22 nt down- primer, and forward primers that annealed within specific genes stream of the translation termination codon (Fig. 2 and Fig. S2). (Fig. 1 and Dataset S1). All five K. mikimotoi chloroplast tran- In the case of psbA, a single consensus poly(U) site was observed scripts tested (psbA, psbC, psbD, psaA,andrbcL)gavePCR in every sequenced clone, whereas the precise poly(U) site varied products of the expected sizes, indicating that the corresponding by up to 5 nt in all other genes (Fig. 2). To verify that the poly(U) transcripts were indeed polyuridylylated (Fig. 1, lanes 1–5). We tracts observed were added posttranscriptionally, we amplified the confirmed this by sequencing (see

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