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Home , Chloroplast

Chloroplast Origin and Integration1

Geoffrey I. McFadden* Research Centre, School of , University of Melbourne, Victoria 3010, Australia

The origin of oxygenic must rank lope (12, 13). It has subsequently emerged that just after the origin of itself as one of the most (and probably ) have learned to operate these significant events in the . The early transporters in reverse to “feed” their at photosynthesizers, which Bill Schopf has shown night or in organs underground. Norman Weeden were similar to modern (32), made then recognized that the , in addition earth a nicer place to live by quietly cranking out to the gift of photosynthesis, was responsible for the over hundreds of millions of years. Having introgression of several other metabolic pathways established an ozone UV shield, the cyanobacteria into the host (38). Weeden’s insight was the spur for then entered into an extraordinary partnership with the ongoing characterization of the bacterial-type eukaryotic cells: They became chloroplasts. Equipped pathways for amino biosynthesis, with chloroplasts, the early plants were ready to biosynthesis, non-Shemin tetrapyrole bio- colonize the land and the planet. This review synthesis, non-mevalonate isoprenoid biosynthesis, recounts major leaps in our understanding of chloro- and nitrate and sulfate assimilation. Weeden also plast from the preceding 25 years. recognized that many of the for these processes must have undergone intracellular transfer from to nucleus. ENDOSYMBIOSIS TRIUMPHS ONE RING TO RULE THEM...AND IN THE In 1975 the theory of endosymbiosis (which de- DARKNESS BIND THEM scribes the origins of mitochondria and from eubacteria-like cells living within eukaryotic hosts) The advent of Sanger-style DNA sequencing was still hanging in the balance. Major players like opened the way to more ambitious projects. Oyha- Lawrence Bogorad (4) and Tom Cavalier-Smith (7) ma’s team (28) was first to sequence a chloroplast were weighing the merits of the autogenous (nonen- determining the 121,024 bp of the liverwort dosymbiotic) alternatives for chloroplasts against the Marchantia polymorpha chloroplast. The Japanese early compelling scenarios of Mereschkowsky and group’s work inspired numerous other chloroplast later . However, in the same year, an genome sequencing projects from which various pat- innovative paper from Linda Bonen and Ford terns, and a few surprises, have emerged. The March- Doolittle (5; which provided the first quantitative antia genome has proven to be a good general model. measure of similarities between T1 rRNA catalogues Typical chloroplast , including those of plants of cyanobacteria and chloroplasts) is now recogniz- and most algae, are circular with genes organized in able as the first ripple in a tidal wave of chloroplast . Plastid content varies from about 70 in molecular data that swept the autogenous origin hy- some non-photosynthetic plastids such as that of the pothesis away. parasite (39) up to about 200 in algae (30); Hans Ko¨ssel was one of the first to apply the re- plants typically have around 100 genes in the chlo- cently developed Maxan/Gilbert DNA sequencing roplast. algal chloroplasts are the only technology to chloroplasts. Primary and secondary example that so far seriously defy this trend, having structure of maize chloroplast rRNA was revealed to single gene mini-circles that encode only a handful of be more closely related to the bacterium Escherichia genes (40). One ring no longer binds them. coli than to equivalent genes in the nuclei of eu- The next major leap in the application of sequenc- karyotes (the yeast sequence only became available ing was the complete genome of the cyanobacterium to Ko¨ssel at the proof stage!), corroborating the en- of Synechocystis (20). The Japanese team identified dosymbiotic hypothesis (34). 3,229 genes, which provided a reference point for the Flu¨ gge and Heldt recognized that integration of the gene content of the chloroplast endosymbiont at the endosymbiont required a transporter for export of outset of endosymbiosis. For the first time we held a photosynthate and characterized a family of phos- list of genes with which the endosymbiont probably phate translocators located in the chloroplast enve- started. Comparisons of this list with the present content of plastid issues some weighty 1 G.I.M. is supported by the Australian Research Council and is challenges. an International Scholar of the Howard Hughes Medical Institute. The genome sequences also provided an opportu- * E-mail [email protected]; fax 61–3–9347–1071. nity to compare gene synteny between chloroplasts

50 , January 2001, Vol. 125, pp. 50–53, www.plantphysiol.org © 2001 American Society of Plant Physiologists Chloroplast Origin and Integration and . Conservation of large operons proved within the chloroplast prior to assembly of the ho- that chloroplast DNAs are reduced bacterial ge- loenzyme. Gregory Schmidt and colleagues (31) de- nomes (28). In a converse manner, unique gene as- termined the leader sequence and the site of process- semblages shared among all chloroplasts suggest that ing. Chua and Schmidt (9) and Highfield and Ellis all chloroplasts arose from a single primary endo- (19) subsequently demonstrated that the transloca- (30), as first argued by Tom Cavalier-Smith tion into the chloroplast occurs post-translationally. (8). Many other studies now concur, but the idea is These experiments were important for two key rea- not universally accepted (23). Large amounts of se- sons: first, they showed that chloroplast targeting quence data have also allowed meta-analyses in was different to the “signal hypothesis” for cotrans- which multiple genes are used in determining more lational insertion into the , robust phylogenies (25). These are consistent and second, they were the germ of a system to dissect with the belief that the alga Cyanophora, the import process. By using in vitro translated pre- which has a blue-green chloroplast with a pepti- cursors in conjunction with isolated plastids it has doglycan wall, is one of the earliest diverging lines to been possible to manipulate the system experimen- contain a chloroplast. Meta-analysis also confirms tally to identify conditions required for transport and morphological clues suggesting that the prasino- components of the transport machinery. Mishkind et phyte Mesostigma is the closest living relative to the al. (27) demonstrated that the transit was unicellular alga from which the land plants are de- sufficient and necessary for import of . scended (22). Schrier et al. (33) and van den Broek et al. (37) estab- lished the use of transit to direct foreign proteins into chloroplasts in transgenic plants, which INTRACELLULAR GENE TRANSFER AND paved the way for targeting of reporter proteins such TARGETING as the jellyfish green fluorescent protein. A key development in understanding the mecha- An important outcome of comparing the gene con- nisms of import was the identification of ATP as the tent of a cyanobacterium with that of chloroplasts is essential requirement for transport (16). This depen- the detailed reconstruction of genome reduction. dence was exploited to interrupt transfer, which en- When it was first established that nuclear genes en- abled the partially transferred precursor to be used as code some types of chloroplast proteins it became a tag (often through innovative use of cross linkers or clear that chloroplasts were genetically only semi- autonomous and had relinquished genes to their affinity motifs attached to the targeted protein) to hosts. The genome maps in conjunction with the recover components of the transport apparatus. Ap- phylogeny tell us that this process occurred in a proaches based on this strategy have identified nu- somewhat ad hoc manner, although certain classes of merous transport-related proteins (known as Tocs genes are apparently less amenable to transfer (25). and Tics) located in the inner and outer chloroplast Two intriguing questions are how many nuclear membranes such as the receptors (36) and the chan- genes service the chloroplast and are they all derived nel protein (24). from the endosymbiont cyanobacterium. The Arabi- The targeting of proteins within the confines of the dopsis is beginning to allow us to chloroplast was first studied by Smeekens et al. (35). address these questions; early surveys indicate that They demonstrated that was targeted as many as 2,000 nuclear genes encode proteins with into the chloroplast by a typical transit pep- plastid functions (1). The tagged mutagenesis pro- tide, but that a second signal, this time homologous gram also provides a means to assay the functions of to bacterial peptides, directed the protein those genes whose products are predicted to be chlo- across the membranes. In other words, the roplast targeted, but for which no obvious function is original bacterial mechanisms for targeting proteins apparent. across the inner membrane have been retained in The transfer of genes from chloroplast to nucleus chloroplasts, a phenomenon now termed “conserva- typically requires return passage of the gene product tive sorting.” The transit peptide was thus perceived to its place of function. This mechanism is central to as a prefix appended to the protein to return it to the the establishment and refinement of endosymbiosis. for internal distribution. The first vagrant gene to be studied was the Rubisco The intense study of Rubisco allowed plant biolo- small subunit (RbcS), and it was established early gists to discover a major unifying principle for the that a precursor form was produced in the assembly of oligomeric protein structures. John Ellis and targeted. It was subsequently established that and colleagues (3) identified a 60-kD protein essential the precursor bears an N-terminal extension, the so- for Rubisco assembly. Sequencing of the so-called called “transit peptide,” which mediates transport. “Rubisco-binding protein” and the GroEL heat shock There were several key developments in the under- proteins of E. coli demonstrated that these proteins, standing of this process. Dobberstein et al. (10) estab- which Ellis dubbed chaperones, are fundamental lished that RbcS began as a precursor with a small and ubiquitous components of and (3.5 kD) extension that is proteolytically removed protein/protein interactions (18). This discovery is

Plant Physiol. Vol. 125, 2001 51 McFadden one of the rare instances where plant science forged of chloroplast gene (17). The dual tran- the way into a unifying concept with application scription systems are thought to allow regulation of across all . chloroplast development—a hypothesis whose test- ing will require further transgenics. The second major development stemming from SECOND-HAND CHLOROPLASTS chloroplast transformation was the laboratory recon- struction of intracellular gene relocation. Pal Maliga’s Plant chloroplasts have two bounding membranes, group was able to delete the chloroplast gene for but chloroplasts of many algae have three or four RbcL and complement the deletion by inserting a membranes. Sally Gibbs (15) first articulated the idea nuclear copy, complete with a motif to target the that these multi-membraned, or complex, chloro- product, into the chloroplast (21). Perhaps the great- plasts were acquired indirectly, not by the classic est promise for chloroplast transformation lies in mechanism of endosymbiosis of a cyanobacterial-like commercial applications (14). The chloroplast ge- . Gibbs proposed that the chloro- nome has many features that make it an ideal site for plast, for instance, derived from a eukaryotic alga insertion of useful genes. Chloroplast DNA is multi- that had been engulfed by a eukaryotic phagotroph copy per cell, which ensures a high gene dosage and (15). The engulfed cell underwent drastic reduction generally high expression levels for . Un- such that in most cases the only residues are the like nuclear plant transformation, genes can be tar- chloroplast and the extra membranes created by the geted to specific sites in chloroplasts. This precision engulfment. Acquisition of chloroplasts in this man- avoids the poorly understood “position effect” ner occurred multiple times (the number of acquisi- whereby the site of insertion of transgenes influences tions is argued hotly) and thus explains the patchy behavior in an unpredictable manner. distribution of chloroplasts across the eukaryotic Chloroplast transgenics makes genetic engineering of . The process was first verified in plants more controlled. Last, because chloroplasts are where a minute residue of the endosymbiont nucleus maternally inherited in angiosperms, the risk of has been identified (11). This process of acquisition is transgene spread into the environment through pol- known as secondary endosymbiosis, as distinct from len is greatly reduced. primary endosymbiosis between a host and a prokaryote endosymbiont. Secondary endo- symbiosis allowed lateral transfers of chloroplasts THE FUTURE into non-photosynthetic lineages, including unex- pected lineages such as the malaria parasite (26, 39). The breakthroughs outlined here position us to Gibbs also predicted (correctly) how proteins could tackle some fundamental questions in the next 25 be targeted across the multiple bounding membranes years. We will soon have full gene complements for by initial utilization of the secretory pathway to cross the host and endosymbiont and the ability to manip- the outermost membrane. ulate both genomes and target foreign proteins from the host to the endosymbiont (there are no leads on mechanisms to do the reverse). These approaches will REFORGING THE RING be central in developing our understanding of still mysterious processes such as chloroplast division (29), During its hundreds of millions of years tenure the molecular signals regulating plastid differentia- within the host, the chloroplast genome has under- tion, and the mechanisms of cross talk between the gone substantial modification. Human intervention and its little green slaves. Armed with this is now beginning to make even more drastic modifi- information we may be in a position to undertake cations to chloroplast DNA. Transgenics is an im- some very bold experiments. We may even be able to mensely powerful tool for biological understanding. reconstruct endosymbiosis in the laboratory, putting Chloroplast transformation was first achieved in the chloroplasts into non-photosynthetic hosts. green alga using a “gene gun” ap- proach in which selectable markers were literally blasted into the cells (6). However, engineering of ACKNOWLEDGMENTS chloroplast genomes has not really achieved wide- spread application. Nevertheless, chloroplast trans- Apologies to those whose work I could not cite. Thanks formation has opened doors to some exciting devel- to Bill Martin who shared his literature knowledge so opments. Abrogation of the chloroplast’s indigenous enthusiastically. transcription system (knockouts of a cyanobacterial- like rpo gene in chloroplast DNA) provided definitive LITERATURE CITED evidence for the existence of an elusive alternate polymerase (2). A single subunit, phage-type poly- 1. Abdallah F, Salamini F, Leister D (2000) Trends Plant merase, encoded by the nucleus and similar to that Sci 5: 141–142 used for mitochondrial transcription, was subse- 2. Allison LA, Simon LD, Maliga P (1996) EMBO J 15: quently shown to be responsible for a major fraction 2802–2809

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