PP66CH03-Bock ARI 18 November 2014 12:43 R E V I E W S I N A D V A N C E Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology Ralph Bock Max-Planck-Institut f ¨ ur Molekulare Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany; email: [email protected]Annu. Rev. Plant Biol. 2015. 66:3.1–3.31 The Annual Review of Plant Biology is online at plant.annualreviews.org This article’s doi: 10.1146/annurev-arplant-050213-040212 Copyright c 2015 by Annual Reviews. All rights reserved Keywords plastid transformation, chloroplast transformation, reverse genetics, metabolic engineering, molecular farming, experimental evolution, horizontal gene transfer Abstract The small bacterial-type genome of the plastid (chloroplast) can be engi- neered by genetic transformation, generating cells and plants with transgenic plastid genomes, also referred to as transplastomic plants. The transforma- tion process relies on homologous recombination, thereby facilitating the site-specific alteration of endogenous plastid genes as well as the precisely targeted insertion of foreign genes into the plastid DNA. The technology has been used extensively to analyze chloroplast gene functions and study plastid gene expression at all levels in vivo. Over the years, a large toolbox has been assembled that is now nearly comparable to the techniques avail- able for plant nuclear transformation and that has enabled new applications of transplastomic technology in basic and applied research. This review de- scribes the state of the art in engineering the plastid genomes of algae and land plants (Embryophyta). It provides an overview of the existing tools for plastid genome engineering, discusses current technological limitations, and highlights selected applications that demonstrate the immense potential of chloroplast transformation in several key areas of plant biotechnology. 3.1 Review in Advance first posted online on December 1, 2014. (Changes may still occur before final publication online and in print.) Changes may still occur before final publication online and in print Annu. Rev. Plant Biol. 2015.66. Downloaded from www.annualreviews.org Access provided by CONICET on 01/27/15. For personal use only.
The small bacterial-type genome of the plastid (chloroplast) can be engi-neered by genetic transformation, generating cells and plants with transgenicplastid genomes, also referred to as transplastomic plants. The transforma-tion process relies on homologous recombination, thereby facilitating thesite-specific alteration of endogenous plastid genes as well as the preciselytargeted insertion of foreign genes into the plastid DNA. The technologyhas been used extensively to analyze chloroplast gene functions and studyplastid gene expression at all levels in vivo. Over the years, a large toolboxhas been assembled that is now nearly comparable to the techniques avail-able for plant nuclear transformation and that has enabled new applicationsof transplastomic technology in basic and applied research. This review de-scribes the state of the art in engineering the plastid genomes of algae andland plants (Embryophyta). It provides an overview of the existing tools forplastid genome engineering, discusses current technological limitations, andhighlights selected applications that demonstrate the immense potential ofchloroplast transformation in several key areas of plant biotechnology.
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Review in Advance first posted online on December 1, 2014. (Changes may still occur before final publication online and in print.)
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The chloroplasts of all eukaryotic algae and all embyrophytes (bryophytes, ferns, and seed plants)are the product of a singular endosymbiotic event that happened approximately 1.5 billion years agoand occurred through the uptake of a cyanobacterium by a heterotrophic protist. The subsequentgradual evolutionary optimization of the relationship between the host cell and the endosymbiontinvolved several key innovations, including (a) the establishment of metabolite exchange systemsthat facilitate the transport of reduced carbon compounds across the double membrane of theendosymbiont, (b) genome streamlining by elimination of dispensable and redundant geneticinformation, (c) massive gene transfer from the endosymbiont genome to the host nuclear genome,and (d ) the evolution of a protein import machinery that reroutes the gene products of transferredgenes into the chloroplast. The combined action of genome streamlining and gene transfer resultedin a dramatic shrinkage of the genome of the cyanobacterial endosymbiont, in that thousands ofgenes disappeared and were either deleted or moved to the nucleus (23, 139). Consequently,present-day plastid (chloroplast) genomes of photosynthetic eukaryotes are much reduced andtypically harbor only 100–250 genes (approximately 130 genes in seed plants) (Figures 1 and 2).From its structure and sequence, the genome is still clearly recognizable as a remnant of thegenome of the ancestral cyanobacterial endosymbiont (16, 183). The plastid DNA (or plastome)
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Figure 1Physical map of the Chlamydomonas reinhardtii chloroplast genome, drawn using the complete genome sequence as input (GenBankaccession number NC_005353.1) in version 1.1 of the OrganellarGenomeDRAW software tool (106, 107). The gray arrows denote thedirection of transcription for the two DNA strands of the (circularly mapping) genome, and the interior circle shows its tetrapartitestructure. Abbreviations: IRA, inverted repeat A; IRB, inverted repeat B; LSC, large single-copy region; SSC, small single-copy region.
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Embryophyta:a subkingdom of greenplants (Plantae) thatcomprises bryophytes,ferns and their allies,and seed plants
Mesophyll:parenchyma cells(L2 + L3 layers) thatlie between the upperand lower epidermis(L1 layer) of the leaf
of most embryophyte plants and green algae shows a tetrapartite genome organization, with alarge single-copy region (LSC) and a small single-copy region (SSC) separating two invertedrepeat regions. The two inverted repeats are identical in their nucleotide sequence and differ onlyin their relative orientation (Figures 1 and 2).
The availability of technology for chloroplast genome engineering led to a major upsurge inresearch on chloroplast genomes, gene functions, and gene expression. This article describes themethodology of plastid transformation and the toolbox that has been assembled by the communityover the years, discusses current limitations and future challenges in plastid genome engineering,and provides an overview of applications of the technology in basic research and biotechnology.
PLASTID TRANSFORMATION METHODS FOR ALGAE AND PLANTS
The key innovation that made organelle transformation possible was the invention of the gene gun,a device that allows researchers to bombard living cells and tissues with accelerated DNA-coveredmicroparticles. The technology became known as biolistic (biological + ballistic) transformation,and because it relies entirely on physical principles, it provides a universal method for introducingnaked (purified or synthetic) nucleic acids into essentially any organism or cell type. Amazingly,the technology works for cell organelles as well, even though they are in the same size range as(chloroplasts) or even smaller than (mitochondria) the standard particle size used for shooting(0.4–1.7 μm).
Transformation of the chloroplast genome was first accomplished in Chlamydomonas reinhardtii,a unicellular green alga harboring a single chloroplast that occupies approximately half the cellvolume and contains approximately 80 identical copies of the plastid genome (27) (Figure 1).Chloroplast transformation was thought to be much more challenging in seed plants (and waseven believed to be impossible by quite a few researchers in the field) because a typical leafmesophyll cell contains 1,000–2,000 copies of the plastid genome (Figure 2) and approximately100 chloroplasts (68). However, soon after the initial success with Chlamydomonas, chloroplasttransformation was achieved in tobacco (Nicotiana tabacum) (169). For many years, Chlamydomonasand tobacco remained the only two species that were routinely transformable (Tables 1 and 2).In these two model systems, the basic principles of plastid genome engineering were worked out,essentially all currently available tools were developed, and most proof-of-concept applicationswere conducted.
The genome size, coding capacity, and genome organization of the two model systems forchloroplast transformation are similar but not identical. For example, as a result of recent (lineage-specific) endosymbiotic gene transfer events, a number of genes encoded in the plastid genome oftobacco are encoded in the nuclear genome of Chlamydomonas and vice versa (Figures 1 and 2).
Transformation Methods
Although biolistic transformation has remained the method of choice for plastid transformationin both algae and embryophyte plants (Table 2), a few alternative transformation protocols have
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Physical map of the tobacco (Nicotiana tabacum) plastid genome, drawn using the complete genome sequence as input (GenBankaccession number NC_001879.2) in version 1.1 of the OrganellarGenomeDRAW software tool (106, 107). The gray arrows denote thedirection of transcription for the two DNA strands of the (circularly mapping) genome, and the interior circle shows its tetrapartitestructure. Abbreviations: IRA, inverted repeat A; IRB, inverted repeat B; LSC, large single-copy region; SSC, small single-copy region.
Biolistic bombardment of leaves,PEG treatment of protoplasts
aadA, rRNA alleles 131, 146
Lactuca sativa (lettuce) Spermatophyta,Asteraceae
Biolistic bombardment of leaves,PEG treatment of protoplasts
aadA 87, 101
Species in which no integration of the transforming DNA into the plastid genome could be demonstrated (e.g., the alga Euglena gracilis), stablehomoplasmy could not be achieved (e.g., rice), or no fertile plants could be recovered (e.g., Arabidopsis) have been omitted. Abbreviation: PEG,polyethylene glycol.
Protoplast: a wall-lessplant cell, typicallyproduced by enzymaticremoval of the cell wall
been developed. Typically, they require cell cultures and removal of the cell wall prior to transfor-mation (or, alternatively, use of cell wall–deficient mutant strains), which makes the procedurestechnically more demanding, labor intensive, and time consuming. In Chlamydomonas, agitatingcell wall–deficient cells in the presence of glass beads and transforming plasmid DNA can resultin chloroplast transformation (54). Similarly, in multicellular plants, incubation of isolated proto-plasts with the polyether polyethylene glycol (PEG) and plasmid DNA allows selection of stablechloroplast transformants (Table 2). In both cases, DNA uptake is likely to be facilitated by theclose proximity of chloroplasts to the plasma membrane, which may allow the passage of DNAthrough three tightly appressed layers of membranes if their structure is sufficiently loosened byphysical or chemical means. In multicellular plants, PEG-mediated plastid transformation requiressubsequent regeneration of plants from (wall-less) protoplasts, which is a sensitive and lengthyprocess that is not well established for many plant species.
In summary, biolistics is at present unrivaled in both speed and transformation efficiency.Although alternatives to the biolistic protocol are available, unless the costs of the instrumentationand/or intellectual property issues are a serious consideration, there is currently no compellingreason to move away from particle gun–mediated chloroplast transformation.
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Aminoglycoside: aclass of antibiotics thatcontain an amino-modified glycoside(sugar) and act asinhibitors of bacterialprotein biosynthesis
Polyploidy: the stateof possessing morethan two completecopies of the geneticinformation in a cell ora genetic compartment
Homologousrecombination:reciprocal exchange ofnucleotide sequencesbetween two similar oridentical DNAmolecules
Selectable Markers
Table 1 provides an overview of established selectable marker genes for primary selection oftransplastomic cells. Historically, chloroplast transformation was developed by using endogenouschloroplast sequences as selectable markers: a photosynthesis-related gene that restores photoau-totrophic growth in Chlamydomonas, and point mutations in the chloroplast 16S rRNA that conferantibiotic insensitivity to plastid translation in tobacco (Table 1). However, especially in seedplants, these markers quickly fell out of fashion when much more efficient transgene-based se-lectable markers were developed. A single antibiotic resistance marker, initially developed forChlamydomonas and subsequently adapted for tobacco (70, 170), turned out to be a lucky strike:the aadA gene from the gut bacterium Escherichia coli. This gene encodes an aminoglycoside3′′-adenylyltransferase, an enzyme that inactivates several antibiotics of the aminoglycoside typethrough covalent modification (i.e., attachment of an AMP residue). Importantly, spectinomycinand streptomycin, two aminoglycoside antibiotics that act as potent inhibitors of plastid trans-lation, are efficient substrates of the AadA enzyme. Spectinomycin selection turned out to beparticularly effective because of the high specificity of the drug to the chloroplast 70S ribosomeand its low mutagenic and other side effects (Figure 3). Although several alternative selectablemarker genes have been developed over the years (Table 1), the aadA gene is still superior to all ofthem. Besides the high specificity of spectinomycin as an inhibitor of plastid translation, the highenzymatic activity of the AadA protein is likely to also contribute to the unparalleled efficiency ofthe aadA marker gene in combination with spectinomycin selection.
In addition to positive selectable markers that facilitate the selection of transplastomic cells, afew negative selectable marker genes have been established in chloroplasts (66, 151). These conferconditional lethality and are likely to become useful in genetic screens for regulators of plastidgene expression.
Integration of Foreign DNA into the Plastid Genome
Stable transformation of chloroplasts requires (a) integration of the transforming DNA into theresident plastid DNA and (b) elimination of all untransformed copies of the highly polyploidplastid genome. Integration of foreign DNA into the plastid genome appears to occur exclusivelyby homologous recombination. This is reflected in the design of vectors for plastid transformation:The vectors must contain flanking regions of homology to the targeted integration site in theplastid genome (Figure 4). The efficiency of targeting is positively correlated to the length ofthese flanking regions (41), but it is generally believed that flank sizes of 0.5–1 kb are sufficient andthat the transformation frequency does not increase much if larger flanks are used. Homologousrecombination in chloroplasts is remarkably efficient, and usually only the final recombinationproducts resulting from double-crossover events are detectable (Figure 4).
A specific challenge associated with chloroplast transformation lies in the high degree of poly-ploidy of the plastid genome, with up to 1,000 copies or more of the plastid DNA being presentin a single cell. It is generally assumed that the primary transformation event changes only one(or at most a few) genome copies. Consequently, transplastomic cells are initially heteroplasmicand contain a mixed population of wild-type genome copies and transformed genome copies. Thegenomes segregate freely upon subsequent rounds of cell division and organelle division. In theabsence of selection, heteroplasmy of randomly segregating DNA molecules is genetically un-stable, and sooner or later homoplasmic cells (harboring only one of the two genome types) willarise. To prevent the loss of the transgenic plastid genome (or transplastome), transplastomic celllines are continuously propagated under selection pressure until all residual wild-type genomes are
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a b c
d e f
Figure 3Generation of transplastomic potato (Solanum tuberosum) plants. (a) Preparation of leaves for biolistictransformation. Young leaves of potato plants grown under aseptic conditions are arranged to cover thesurface of a standard petri dish. (b) Exposure of biolistically bombarded leaf explants to a spectinomycin-containing regeneration medium. (c) Selection of primary transplastomic lines. After 11 weeks of selectionon spectinomycin-containing medium, the leaf explants are swollen and largely bleached owing to inhibitionof chloroplast protein biosynthesis. The arrow points to a putative transplastomic clone that is resistant tospectinomycin and regenerates into plantlets. (d ) Additional regeneration round under spectinomycinselection to purify the transplastomic line to homoplasmy. Leaflets from regenerating plantlets of theprevious regeneration round were exposed to a spectinomycin-containing plant regeneration medium andregenerated again into shoots (picture taken after 11 weeks). (e) Additional regeneration round initiated fromstem sections. The efficiency of regeneration and purification to homoplasmy from stem explants is similarto that from leaf explants (picture taken after 11 weeks). ( f ) Growth of homoplasmic transplastomic plantsunder aseptic conditions on a synthetic medium. Note the development of microtubers from the roots.
Gene conversion: anonreciprocal transferof genetic informationin which one DNAsequence replaces ahomologous DNAsequence such that thetwo sequences becomeidentical
eliminated. In seed plants, the homoplasmic transplastomic state is typically achieved after two orthree additional rounds of plant regeneration under selection (i.e., in the presence of spectinomycinif aadA was used as the selectable marker gene) (15, 116).
If the gene or mutation to be introduced into the plastid genome is not absolutely linkedto the selectable marker gene (Figure 4b,c), extended periods of heteroplasmy bear the risk ofgene conversion between the two genome types (89). Gene conversion can eliminate the unlinkedmutation so that, in the end, genomes harboring only the selectable marker gene but not thedesired mutation or transgene are obtained, even though initially the desired change was there. Itis therefore of the utmost importance to (a) pass the transplastomic lines through the additionalselection cycles quickly to bring them to the stable homoplasmic state as fast as possible, and(b) analyze many regenerants in each regeneration round (dozens or sometimes hundreds) bySouthern blotting and/or amplification by polymerase chain reaction (PCR) and sequencing. Thelatter is particularly important to be able to act quickly upon seeing the first signs of gene conversion(by checking many more regenerants or by going back to the previous regeneration round).
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ptDNA
Transformation vector
aadA
Left flank aadA
ptDNA
Transformation vector
aadA
Knockout• Gene disruption
• Gene deletion (replacement)
Site-directed mutagenesis• Introduction of point mutations,
insertions/deletions, etc.
Cotransformation• Gene knockout
• Site-directed mutagenesis
a b c
Transformed ptDNATransformed ptDNA
Right flank
Target gene
Target gene
Left flank aadARight flank
Target gene
Target gene
M
M
Left flank
aadA
ptDNA
Transformation vector I
aadA
Cotransformed ptDNA
Right flank
Target gene
Target gene
M
M
Transformationvector II
Leftflank
Rightflank
Figure 4Strategies for modifying endogenous genes in the chloroplast genome [plastid DNA (ptDNA)]. (a) Construction of a gene knockout bydisruption of the reading frame with the selectable marker gene cassette (aadA; red box). Alternatively, the target gene ( green box) can beexcised from a cloned ptDNA fragment and replaced with the marker cassette. “Left flank” and “right flank” denote flanking regions ofhomology in which homologous recombination can take place (blue boxes). Possible recombination events (double crossovers) leading tosuccessful plastid transformation are indicated by dashed arrows. (b) Introduction of mutations into a plastid gene by site-directedmutagenesis. Note that recombination in the region between the mutation (M) and the aadA gene ( gray dashed arrow) results inuncoupling of the two genetic changes and, hence, produces transplastomic lines that harbor the selectable marker gene but not thedesired mutation in the target gene. The frequency of appearance of such transplastomic lines depends on the distance of the mutationfrom the aadA marker relative to the size of the left flank. (c) Introduction of mutations into a plastid gene by cotransformation. If thetarget gene is embedded in a complex operon, it may not be possible to insert the aadA marker in close proximity without interferingwith the expression of other genes in the operon. In these cases, the aadA gene can be incorporated into a separate transformationplasmid (transformation vector II) and targeted to a neutral region elsewhere in the genome. Cointegration of the aadA gene and the(unselected) mutation in the target gene (supplied on transformation vector I) will occur in 5–20% of the transplastomic clones (95).
A common error in the analysis of transplastomic lines is to mistake promiscuous plastid DNAin the nucleus or the mitochondrion for heteroplasmy. Weak wild-type-like hybridization signalsin DNA gel blot analyses or wild-type-like bands in PCR assays that persist over the regenerationrounds often come from plastid DNA transferred to the nuclear or mitochondrial genome. Inthese cases, homoplasmy can be verified by Southern blots with purified plastid DNA and/orby crosses and segregation assays that demonstrate a lack of phenotypic segregation in the nextgeneration (73, 96).
Cotransformation
Particle bombardment allows the simultaneous transformation of cells with multiple vectors.Although one might think that plastid transformation is so inefficient that the recipient chloroplast
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Episome: a DNAelement that is notincorporated into thegenome and canreplicateautonomously
usually takes up only a single plasmid molecule, successful cotransformation with two plasmidshas been demonstrated in both Chlamydomonas and tobacco (31, 91). Importantly, recovery ofcotransformants does not require double selection for both transformation plasmids, thus allowingtargeting of the selectable marker gene to one region of the plastid genome while introducing agenetic change (or another transgene) into a totally different region of the genome (Figure 4c).
The frequency of cointegration of the unselected transgene or mutation is somewhat variable.Cotransformation frequencies between 5% and 20% have been reported in tobacco (31, 95),and it seems possible that mutations entailing negative phenotypic consequences result in lowfrequencies. Cotransformation approaches are particularly useful if defined changes are to beintroduced into large, complex operons, where linkage to the selectable marker gene is not possibleor bears the risk of interfering with transcription and/or RNA processing (95) (Figure 4c).
Surprisingly, it is also possible to cotransform the nucleus and the plastid at the same time(58). If particles are simultaneously coated with a vector for nuclear transformation and a vec-tor for plastid transformation, doubly transformed (transgenic and transplastomic) cells can berecovered. This type of cotransformation seems to require the presence of both plasmids on thesame particle, because mixing particles that were individually coated with the two plasmids didnot result in plastid-nucleus cotransformation. These findings have interesting implications re-garding the mechanisms of biolistic DNA delivery and DNA uptake by the target compartment(58).
Transformable Species and Bottlenecks in Extending the Species Range
After the initial success with plastid transformation in Chlamydomonas and tobacco, progress withextending the species range of the technology has been disappointingly slow. Table 2 lists speciesfor which plastid transformation has been reproducibly obtained and confirmed by at least twoindependent reports. More than 25 years after the first report of plastid transformation in Chlamy-domonas, the list of transformable species is still very short. Why is that so, and what are thebottlenecks in adapting chloroplast transformation protocols for new species?
Plastid transformation is dependent on (a) a robust method of DNA delivery into the chloro-plast, (b) the presence of an active homologous recombination machinery in the plastid, and(c) the availability of highly efficient selection and regeneration protocols for transplastomic cells.Although switching to a new species may require some optimization of the parameters of thebiolistic procedure (especially adjustment of the particle velocity to the hardness and thickness ofthe cell wall and/or the leaf cuticle to penetrate), given the universality of particle gun–mediatedtransformation, it is generally assumed that the efficiency of DNA delivery does not pose a seriousbottleneck. Homologous recombination is known to be very efficient in Chlamydomonas and seedplant plastids, but there is some uncertainty about its activity in other taxa. For example, in theunicellular flagellate Euglena gracilis, chloroplast transformation could be achieved, but the trans-forming DNA did not integrate into the resident genome and, hence, could only be maintainedepisomally by antibiotic selection (46).
At least in seed plants, where biolistic bombardment requires little species-specific optimization(or is already established for transformation of the nuclear genome) and homologous recombina-tion activity in the plastid is unlikely to be limiting, the efficiency of the tissue culture, selection,and regeneration procedures is considered the most serious bottleneck to plastid transformation.In addition to being easy to grow in tissue culture, tobacco has the great advantage of remain-ing somatically diploid, theoretically allowing plant regeneration from every single cell. Mostother plants undergo somatic endopolyploidization or irreversible cell differentiation during leafdevelopment, making it very difficult to select transplastomic cell lines and/or regenerate fertile
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Callus: anunorganized growingand dividing mass ofplant cells whoseformation in vitro canbe induced bytreatment of tissueexplants withphytohormones
Cybrid: a eukaryoticcell produced by thefusion of a whole cellwith an enucleated cell(cytoplast)
transplastomic plants (157). Therefore, success with plastid transformation in seed plants has beenlimited largely to species for which facile cell and tissue culture systems are available (Table 2).
As described above, spectinomycin resistance conferred by chimeric aadA genes presentlyrepresents by far the most efficient selection principle for transplastomic cell lines (Table 1).Unfortunately, not all plant and algal species are equally sensitive to spectinomycin, and somespecies are even entirely insensitive to the drug. For example, a point mutation that is known toconfer spectinomycin resistance in tobacco is naturally present in the plastid 16S rRNA genes ofall cereals. Other inhibitors of plastid translation, such as streptomycin and kanamycin, do notprovide good alternatives, because they do not sufficiently strongly inhibit callus growth in thedark (104). This is unfortunate, because the most efficient (nuclear) transformation protocols forcereals rely on bombardment of callus propagated in the dark, and selection of transgenic cell linesis conducted in the dark (to prevent terminal cell differentiation). Thus, although biolistic nucleartransformation is routine in many cereals, transformation of the plastid genome is likely to requirenovel selectable markers and/or the development of entirely new tissue culture and regenerationprotocols.
In conclusion, similar to plant nuclear transformation, there is no universal protocol for plastidtransformation. Development of a plastid transformation protocol for a new species represents asignificant challenge that involves tedious optimization work, especially with respect to the tissueculture and selection procedures involved. Unfortunately, this optimization work is exceedinglylaborious and time consuming and is based largely on the trial-and-error principle.
Transfer of Transgenic Plastids Between Species
An alternative to developing a plastid transformation protocol for a new species would be to trans-fer the transgenic chloroplasts from an easy-to-transform species into cells of a nontransformablespecies. A technically challenging way to achieve this involves generating cybrids (cytoplasmichybrids) by using protoplast fusion techniques. To transfer transgenic chloroplasts from a trans-formable into a recalcitrant species, protoplasts must be prepared from both species. The nucleargenome in the protoplasts of the transformable species then needs to be destroyed (for example,by X-ray or γ-ray irradiation). Heterologous fusion of protoplasts from the two species producescells with the nuclear genome of the recalcitrant species and the chloroplast (and mitochondrial)genomes of both species. Subsequent plant regeneration from fused protoplasts in the presenceof the selection agent that kills nontransgenic chloroplasts gives rise to plants harboring the nu-clear genome of the recalcitrant species and the chloroplast genome of the transformable species.Although proof-of-concept studies have demonstrated that this approach works (97, 135, 156),it is technically demanding and limited in applicability, because for species recalcitrant to plastidtransformation, sufficiently efficient protoplast isolation, fusion, and regeneration protocols areusually not available.
The recent discovery that chloroplast DNA moves from cell to cell in tissue grafts (162) openedup the exciting possibility of exploiting this process for the transfer of plastid transgenes betweenspecies. Compared with protoplast fusion and regeneration techniques, plant regeneration fromexcised graft sites is fast and simple (in that it requires only explant exposure to a selective re-generation medium) and is therefore available for many more plant species. The movement ofplastid DNA across graft sites was initially demonstrated for two tobacco cultivars (162) and wassubsequently shown to also occur between different species (164, 173). Transfer of DNA betweengrafted plants is an asexual process and, thus, represents a form of horizontal gene transfer (lateralgene transfer). Analysis of the transferred DNA sequences revealed that entire genomes and pre-sumably entire organelles are transferred, qualifying this process as horizontal genome transfer
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RNA editing:a posttranscriptionalRNA processing stepleading to thealteration of specificnucleotides in anmRNA molecule (inchloroplasts of seedplants by C-to-Uconversion)
(65, 162, 164). Because grafting is not restricted by species boundaries, it can potentially be usedto transfer transgenic plastids even between rather distantly related species. However, the tightcoevolution between the plastid genome and the nuclear genome makes it unlikely that plastidswill function properly when combined with an alien nucleus from a distantly related species (72,150). Therefore, the transfer of transformed plastid genomes between plants will likely remainrestricted to the movement from readily transformable model cultivars used in research to elitecultivars grown commercially, or from a transformable species into a related recalcitrant species(from the same genus or family). Because the molecular determinants of plastome-genome in-compatibilities are still largely unknown, the functionality of a plastid genome transferred into anew host cell cannot be reliably predicted and thus needs to be determined experimentally (72).
THE TOOLBOX FOR PLASTID GENOME ENGINEERING
Over the years, the community has assembled a large toolbox for plastid genome engineering, espe-cially in the two model species Chlamydomonas and tobacco. The following sections briefly reviewthe parts and molecular tools that are relevant to the construction of transformation plasmids.
Vector Backbones
Because integration of foreign DNA into the chloroplast genome relies on homologous recom-bination, there are no universal vectors for plastid transformation. The flanking regions requiredfor targeting (Figure 4) must have sufficiently high sequence homology to the resident plastidgenome to allow efficient homologous recombination. In the plastid genomes of seed plants, genecontent and gene order are well conserved, and the nucleotide substitution rate is 3–4 times lowerthan in the nuclear genomes (49). This high degree of genome conservation usually allows the useof plastid transformation vectors for closely related species (88, 146), avoiding the need to con-struct species-specific vectors. However, a note of caution needs to be sounded here: RNA editingpatterns can differ even between closely related species (85), and heterologous RNA editing sitesoften remain unprocessed when introduced into another species (22, 150). It is, therefore, advis-able to carefully check the sequences of the flanks before using a vector for plastid transformationin a heterologous species.
Numerous vector systems have been developed for plastid transformation in both Chlamy-domonas and tobacco (e.g., 11, 78, 146, 192). Several reviews have discussed frequently used vectorsand considerations for vector choice and vector design (e.g., 17, 110), and I refer interested readersto these articles for more information.
Promoters and Untranslated Regions
A typical expression cassette for plastid transgenes consists of a promoter and a 5′ untranslatedregion (UTR) upstream of the coding region and a 3′ UTR downstream. Promoters recognizedby the plastid-encoded RNA polymerase are of the bacterial type and usually confer significantlyhigher expression levels than the phage-type promoters recognized by the nucleus-encoded RNApolymerase (74). The 5′ UTR contains the ribosome-binding site, also referred to as the Shine-Dalgarno sequence. It engages in complementary base pairing with the 3′ end of the 16S ribosomalRNA and thereby mediates translation initiation. It is important to know that, as in bacteria, thespacing between the Shine-Dalgarno sequence and the initiation codon is absolutely critical to theefficiency of translation initiation in plastids (36, 47, 59). The 3′ UTR confers transcript stability,typically by folding into a stable stem-loop-type RNA secondary structure (165).
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A large number of plastid promoters and UTRs conferring different strengths of transgeneexpression have been described (e.g., 56, 78, 160, 172), including some that confer extraordinarilyhigh transgene expression levels. Owing to the prevalence of translational regulation in plastids(53), the choice of the 5′ UTR is of particular importance. Interestingly, the 5′ UTR from gene 10of bacteriophage T7 proved to be superior to all plastid 5′ UTRs and confers extreme transgeneexpression levels of up to 70% of the total soluble protein in tobacco (98, 132, 186). For reasons thatare not entirely clear, the protein accumulation levels attainable in Chlamydomonas chloroplastsare much lower: Expression levels in the 1–5% range are reached only in rare cases and upon useof nonphotosynthetic mutant strains (117).
An important general consideration in the choice of expression elements is that the repeateduse of identical elements should be avoided, especially if they are larger than 200–300 base pairs.Using several copies of the same element in a directly repeated orientation is particularly dangerousbecause homologous recombination between them induces deletions (84), and indirectly repeatedcopies can cause inversions in the genome (also referred to as flip-flop recombination) (144). Arecent comprehensive review article has summarized all factors known to affect the level of plastidtransgene expression and some resulting general guidelines for vector design (17).
Reporter Genes
Several reporter genes suitable for monitoring gene expression (e.g., in promoter-reporter genefusions) have been adapted for expression in chloroplasts of Chlamydomonas and seed plants. Theseinclude β-glucuronidase (14, 159), luciferases (118, 122), and the green fluorescent protein (GFP)and its derivatives with modified fluorescence properties (28, 142, 155). Especially in seed plants,GFP has quickly displaced all other reporters owing to the high expression levels attainable withboth GFP and GFP fusion proteins and the low background fluorescence in most species (29, 126,155). Selected applications that have provided new insights into the mechanisms and regulationof plastid gene expression are discussed below.
Operon Expression
Plastid genes are arranged in gene clusters that are cotranscribed and, by analogy to bacteria, arecalled operons (Figures 1 and 2). The molecular mechanisms of operon expression in plastids areconsiderably more complex than in bacteria. Plastid operons are often transcribed from multiplepromoters (including additional operon-internal promoters) and require extra processing steps,such as intron splicing and mRNA editing. Moreover, in contrast to bacteria, polycistronic primarytranscripts often undergo posttranscriptional cleavage into monocistronic (or oligocistronic) units.A growing body of evidence suggests that, in many cases, intercistronic cleavage is functionallyimportant to ensure efficient translation (47, 80). The reasons for this are not entirely clear, butaberrant RNA secondary structure formation (80) and a striking 5′-to-3′ decline in the efficiencyat which the individual cistrons of a polycistronic RNA are translated (47) may be involved.Although the requirement for intercistronic processing seems to be sequence context dependent(in that some operons are less dependent on it than others), it is advisable to take processing intoconsideration when constructing synthetic operons (109). Intercistronic processing can be inducedby small sequence elements that fold into stem-loop-type RNA secondary structures and can bederived from processing sites in endogenous chloroplast operons. One such element, dubbed theintercistronic expression element, functions in all heterologous sequence contexts tested so farand, moreover, is short enough to be usable in multiple copies within the same synthetic operonwithout inducing unwanted homologous recombination (109, 149, 190).
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Riboswitch: an RNAsensor that regulatesgene expression(positively ornegatively,transcriptionally ortranslationally) inresponse to thebinding of a smallmolecule, typically ametabolite
Chromoplast: aplastid type specializedin the storage of(colored) carotenoids;it occurs, for example,in flowers and fruits
Amyloplast:a colorless plastid typespecialized in thestorage of starch; itoccurs, for example, inroots and tubers
Inducible and Repressible Gene Expression
A substantial number of genes encoded in the plastid genome are essential and, therefore, are notamenable to functional analysis by gene knockout (48, 149) (see below). Therefore, an inducibleknockdown system would be a valuable tool for chloroplast reverse genetics. Also, the high-levelexpression of some transgenes (e.g., genes encoding hydrophobic proteins) has deleterious effectson plastid functions, including photosynthesis (76, 175, 189), which makes a robust system forinducible transgene expression highly desirable.
Although many genes in the plastid genome respond to light at the level of transcription and/ortranslation, and the expression of some genes is turned off efficiently in the dark, the importanceof light as an energy source and trigger of plant developmental processes prohibits the use of lightas a stimulus for inducible or repressible (trans)gene expression in the chloroplast. Unfortunately,no other exogenous or endogenous cues (e.g., environmental factors or metabolites) are knownthat would regulate plastid genes qualitatively in an on/off manner. Therefore, inducible systemsfor plastids need to be constructed from heterologous components. Initial attempts to build suchsystems placed the inducible component in the nucleus, where efficient tools for inducible geneexpression are available. A T7 RNA polymerase gene driven by an inducible promoter in thenucleus confers inducible expression in tobacco plastids if the protein is targeted to the plastidcompartment by a suitable transit peptide and the target (trans)gene in the plastid genome isplaced under the control of the T7 promoter (108, 119). A conceptually similar inducible systemwas developed for Chlamydomonas chloroplasts. Here, the nuclear transgene encodes a chloroplastprotein that specifically binds to the 5′ UTR of the psbD mRNA and is required for stable mRNAaccumulation. Through the use of an inducible expression system in the nucleus, (trans)genescontrolled by the psbD 5′ UTR can be switched on or off at will by adding or removing thechemical inducer (140, 167).
A more ambitious goal is to develop a chloroplast-only inducible expression system. This wouldhave the advantage of not requiring nuclear transgenes that can outcross in field-grown plants and,therefore, represent a potential biosafety concern. The development of chloroplast-only systemshas been attempted in tobacco, and two such systems have been described. One is based on thelac repressor (LacI) from Escherichia coli and isopropyl β-D-1-thiogalactopyranoside (IPTG) as achemical inducer of transcription (125); the other relies on a synthetic riboswitch that is responsiveto theophylline, a simple plant-derived secondary metabolite (179). Presently, both systems aresomewhat limited in their induction range and are not nearly as efficient as inducible expressionsystems in bacteria and in the nuclei of eukaryotes.
Expression in Nongreen Tissues
The chloroplast genome is highly expressed in photosynthetic tissues, and although there are onlyapproximately 100 proteins encoded in the plastid DNA (Figures 1 and 2), their contribution tothe total protein content of green leaves can amount to more than 50%. Because most of the highlyexpressed plastid genes encode components of the photosynthetic apparatus (thylakoid membraneproteins and the large subunit of RuBisCO), the demand for plastid gene expression capacity innonphotosynthetic tissues is dramatically lower. Consequently, the gene expression machinery ismuch less active in nongreen plastid types, such as chromoplasts and amyloplasts (86, 178). Fora long time, it was thought that this problem was impossible to overcome and that high-levelexpression of plastid transgenes could be achieved only in photosynthetically active chloroplasts.However, recent genome-wide analyses of plastid gene expression at the transcriptional and trans-lational levels (transcriptomics and translatomics) identified a small number of plastid genes that
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Site-specificrecombination:a type of geneticexchange that involvesspecializedrecombinationenzymes and specificDNA sequences(recognitionsequences)
remain expressed at the RNA level or at the level of protein synthesis in chromoplasts of tomatofruits and amyloplasts of potato tubers (86, 178). This finding has facilitated the design of chimericexpression elements in which a plastid promoter from a gene that is still actively transcribed innonphotosynthetic plastid types (but may be downregulated at the translational level) is com-bined with a 5′ UTR from an mRNA that is associated with translating ribosomes (polysomes)in nongreen plastids (18). This strategy was highly successful and resulted in the identificationof promoter-UTR combinations that dramatically improved transgene expression levels in roots,tubers, and fruits (29, 177, 188), with the best-performing constructs reaching 1% of the totalfruit protein in tomato.
Marker Excision
For some commercial applications, it may be desirable to remove the selectable marker genefrom the plastid genome after transformation and attainment of homoplasmy. Also, because ofthe superior performance of the aadA marker, its posttransformation removal can be useful tofacilitate its repeated use in supertransformation experiments (i.e., transformation of an alreadytransplastomic plant). Several techniques for marker gene elimination have been developed forboth Chlamydomonas and tobacco. These methods (a) take advantage of the endogenous homolo-gous recombination activity of the plastid to mediate marker gene deletion; (b) utilize site-specificrecombination systems, such as the Cre/loxP system, to induce marker gene excision; or (c) em-ploy cotransformation and genome segregation approaches (38, 62, 84, 92, 185). Methodologicaldetails and specific advantages and disadvantages of each technique have been reviewed recently(43, 112).
PLASTID TRANSFORMATION IN BASIC RESEARCH
The availability of a transformation technology has revolutionized nearly all areas of chloroplastresearch. It facilitated the in vivo analysis of processes in gene expression that previously couldbe studied only in vitro or not at all. Moreover, it made possible new approaches in functionalgenomics and opened up an entirely new field: experimental genome evolution. The sections belowbriefly review selected areas of basic research that have greatly benefited from this technology.Rather than attempting to give a complete account of what has been done, I focus here on generalapproaches and principles
Reverse Genetics
Because mutations in plastid genomes can be neither easily induced nor mapped, the possibility ofusing chloroplast transformation to make specific changes in plastid genes and open reading frames(Figure 4) was particularly exciting. Over the years, nearly all plastid genes have been targetedin Chlamydomonas and/or tobacco plastids (for a complete list, see 149). This work has provideda wealth of new information about plastid gene functions and structure-function relationshipsin chloroplast protein complexes (e.g., photosystems and ribosomes). One of the unexpecteddiscoveries resulting from the reverse genetic analysis of conserved open reading frames was theidentification of a small group of plastid genes that encode photosystem assembly factors (26, 96,148). Their subsequent in vivo tagging facilitated the isolation of additional (nucleus-encoded)components of the photosystem assembly machinery (2, 136) and thereby provided a novel entrypoint into the challenging problem of photosystem biogenesis. Reverse genetic approaches in
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Synthetic lethality:a condition in which acombination ofmutations in two ormore genes leads tothe death of a cell ororganism, whereaseach individualmutation does not
chloroplasts also furthered our understanding of the molecular mechanisms of gene expression,for example, by elucidating the contributions of wobbling and superwobbling to the reading ofthe genetic code (3, 4, 143)
Figure 4 illustrates common strategies in reverse genetics. Gene inactivation is most easilyachieved by insertion of the selectable marker cassette (to disrupt the reading frame) or replacementof the target gene with the selection marker (Figure 4a). More subtle changes (e.g., point mutationsand small insertions or deletions) are introduced by integrating the selectable marker cassette intoan adjacent intergenic spacer that, ideally, provides a neutral insertion site (Figure 4b). Becausethe plastid genome is rather gene dense, it is not always possible to identify such a neutral insertionsite where the selectable marker gene does not interfere with the expression of neighboring genes.In this case, a cotransformation strategy can be employed (Figure 4c). Cotransformation alsorepresents the most suitable strategy for gene knockout if the target gene is embedded in a largeoperon displaying a complex expression pattern (95)
Systematic reverse genetic analyses in the plastid genomes of Chlamydomonas and tobaccorevealed several essential genes. These genes encode components of the gene expression machinery(e.g., most ribosomal proteins and tRNAs) (1, 144) but also a few other functions (e.g., an essentialprotease subunit and the only plastid-encoded protein involved in fatty acid biosynthesis) (94, 114,153). The essentiality of a plastid gene is revealed by the inability to purify transplastomic knockoutlines to homoplasmy. This is evidenced by stable heteroplasmy in the presence of antibioticselection, a situation also referred to as balancing selection, and by rapid loss of the transplastomein the absence of selection (48). The functional analysis of essential plastid genes requires theidentification of hypomorphic mutations (which cause only a partial loss of function; Figure 4b),methods for inducible gene repression (see above) (140), or conditional knockout approaches (e.g.,by inducible gene excision with a site-specific recombinase) (99)
The availability of alternative selectable markers (Table 1) and the development of markerrecycling techniques (see above) also allow the construction of double or triple knockouts and theintroduction of mutations in multiple plastid genes. Double-knockout approaches are particularlyuseful to probe the functions of two nonessential components of a multiprotein complex in orderto reveal possible molecular interactions or synergistic effects. Recently, double-knockout analysishas been applied to plastid genes encoding nonessential ribosomal proteins (63, 145). This workuncovered a striking case of synthetic lethality in that the combined knockout of two nonessentialribosomal protein genes (rpl33 and rps15) resulted in loss of autotrophic growth (55)
In Vivo Analysis of Gene Expression
Investigations into the mechanisms and regulation of gene expression require faithful experimentalsystems that contain all relevant components and accurately reproduce the functional properties ofthe molecular machinery involved. Because the validity of in vitro studies is often questioned, andin vitro systems are not even available for some steps in gene expression, plastid transformationquickly became widely used as a tool for the in vivo analysis of gene expression and its regulationat all levels.
Transgenic approaches are particularly useful to identify and dissect cis-acting elements in-volved in gene expression at the DNA, RNA, or protein level. Cis-acting elements constitutesequence determinants for transcription, RNA metabolism, translation, and protein stability.They can be studied systematically, for example, by serial deletions, stepwise terminal trunca-tions, or scanning point mutageneses. To this end, the candidate cis-elements are typically placedinto a heterologous sequence context and often additionally tethered to a reporter gene, such as
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uidA (encoding β-glucuronidase) or gfp (encoding GFP). Systematic dissection of cis-elements invivo using plastid transformation has been done for nearly all steps in gene expression, includingtranscription (by analyzing promoter architecture) (5, 93), RNA processing and RNA stability (124,190), RNA editing (19, 20, 34, 77), translation (60, 79, 129, 191), and protein half-life (7). Thesein vivo approaches also provided some limited information about trans-acting factors recognizingthese cis-elements. For example, they revealed the existence of site-specific, compartment-specific,and species-specific RNA editing factors that are encoded in the nuclear genome (21, 22, 33, 168).However, the molecular identification of the trans-acting factors involved in plastid gene expres-sion and its regulation requires combining transplastomic technologies with biochemical and/orgenetic approaches. The latter would greatly benefit from progress with plastid transformationin Arabidopsis thaliana. A workable plastid transformation protocol for this model plant wouldallow transplastomic approaches to be combined with the power of Arabidopsis nuclear genetics,for example, by conducting mutant screens in transplastomic lines expressing fusions of plastidexpression elements with reporter genes.
Experimental Evolution
The acquisition of the cyanobacterial endosymbiont that marked the origin of photosyntheticeukaryotes was followed by the large-scale migration of genes from the genome of the endosym-biont to the nuclear genome of the host cell. This process, also known as endosymbiotic genetransfer (EGT), presumably has been active for more than a billion years and is thought to belargely responsible for the dramatic reduction in size and coding capacity that plastid genomeshave experienced (Figures 1 and 2). Circumstantial phylogenetic evidence and the presence ofapparently recently transferred plastid sequences in the nucleus (so-called promiscuous DNA)have suggested that gene transfer from the plastid genome to the nucleus is still ongoing (9, 121).
The possibility of placing antibiotic resistance genes into the plastid genome and developingrigorous selection schemes to visualize their migration to the nucleus enabled experimental evolu-tion approaches to study EGT in real time. Chloroplast transformation with a kanamycin resistancegene fused to a nuclear promoter produced transplastomic lines that are sensitive to kanamycin,because the nuclear (eukaryotic-type) promoter is not recognized by the prokaryotic-type tran-scription machinery of the plastid. Subsequent selection for kanamycin resistance identified eventsin which the kanamycin cassette had migrated to the nucleus, where the promoter is recognizedby RNA polymerase II. These proof-of-concept studies provided direct experimental proof thatEGT is still active and, moreover, revealed an astoundingly high rate of plastid-to-nucleus genetransfer (81, 163). Molecular analyses of integration events in the nucleus determined the sizes ofthe transferred plastid sequences (82) and provided evidence of frequent instability of the nuclearloci resulting from EGT (152). Moreover, refined genetic screens yielded new insights into themolecular mechanisms of EGT, including the demonstration that direct DNA-mediated genetransfer (rather than RNA/cDNA-mediated transfer) represents the prevailing transfer pathway(64) and the identification of molecular events that convert transferred plastid genes into functionalnuclear genes (161).
Another evolutionary process that could be reconstructed experimentally with the help oftransplastomic approaches is organelle capture (chloroplast capture), a puzzling evolutionary phe-nomenon in which organelle genomes are apparently transferred between species. Whereas pre-viously only cytoplasmic substitution following an introgression event had been considered as acapture mechanism, the discovery of plastid transfer across graft junctions provides a straightfor-ward asexual mechanism (by horizontal genome transfer; see above) that might explain at leastsome cases of organelle capture (162, 164, 173).
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PLASTID TRANSFORMATION IN PLANT BIOTECHNOLOGY
To plant biotechnologists, the plastid genome provides an attractive site for the integration oftransgenes. Although plastid transformation is technically more challenging than nuclear transfor-mation, accommodation of the transgene in the plastid genome offers several notable advantages.These lie in (a) the unique precision of the genetic engineering process in plastids resulting fromthe highly efficient homologous recombination system; (b) the absence from plastids of epigenetictransgene silencing mechanisms that interfere with transgene expression and/or durable expres-sion over generations; (c) the possibility of stacking multiple transgenes in synthetic operons (seeabove); (d ) the extraordinarily high expression levels attainable, especially in seed plant plastids; and(e) the increased biosafety resulting from maternal inheritance of the plastid genome in most crops.The latter greatly reduces the probability of outcrossing of transgenes by pollination (147, 171).
In view of these advantages, many biotechnological applications of plastid transformation havebeen explored over the years. Below, I summarize progress in some of the most intensely researchedareas of plastid biotechnology.
Engineering Resistances
The high transgene expression levels obtainable by transgene expression from the plastid genomemake transplastomic technology an attractive choice in resistance engineering, especially in caseswhere the level of resistance is directly correlated to the expression level of the resistance protein.This is, for example, the case with insect resistance conferred by insecticidal proteins from Bacillusthuringiensis (Bt toxins) and herbicide resistance conferred by expression of herbicide-insensitivemetabolic enzymes (44, 120, 186).
Most of the resistances engineered into chloroplasts so far are based on transgenes that had beensuccessfully used in nuclear transformation before. For example, herbicide resistances were ob-tained to glyphosate (by expression of glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphatesynthases) (186); glufosinate (by expression of the detoxifying enzyme phosphinothricin acetyl-transferase) (84, 111); sulcotrione and isoxaflutole (by overexpression of 4-hydroxyphenylpyruvatedioxygenase) (50); imidazolinone, sulfonylurea, and pyrimidinylcarboxylates (by expression ofinsensitive acetolactate synthases) (154); and D-amino acids (by expression of D-amino acidoxidases) (66).
In addition to the often very high protein accumulation levels, the main advantage of resistancegene expression from the plastid genome lies in the increased transgene containment provided bymaternal chloroplast inheritance. Extreme expression levels have been reached in some studies,for example, with Bt toxin genes expressed in tobacco chloroplasts (44). However, in one reportedcase, high-level Bt protein accumulation in transgenic plastids resulted in a growth phenotype(32), suggesting that the expression level of the resistance gene needs to be carefully optimized inorder to provide sufficient protection without incurring a yield penalty.
Metabolic Engineering
A number of studies have been performed to evaluate the potential of transplastomic technologyfor metabolic pathway engineering, with the goal of increasing the nutritional value of cropplants or exploiting plants as production factories for metabolites of commercial interest. Becausethe chloroplast harbors a large number of biosynthetic pathways (and is often referred to asthe biosynthetic center of the plant cell), many biochemical pathways in plants are amenableto engineering via chloroplast transformation. An important restriction is that plastid-producedenzymes stay put and cannot be exported from the organelle. Thus, metabolic pathway engineering
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Subunit vaccine:a vaccine presenting a(protein) antigen tothe immune systemrather than the entirepathogen (viralparticles, bacteria)
through plastid transformation requires the presence of an accessible metabolite pool within thechloroplast.
The feasibility of metabolic engineering in transgenic plastids has been demonstrated for sev-eral nutritionally important biochemical pathways, including carotenoid biosynthesis (6, 75, 184)and fatty acid biosynthesis (39, 113). The possibility of transgene stacking in synthetic operonsarguably represents the greatest attraction of the transplastomic technology for metabolic pathwayengineering (149). Because initial attempts to express native bacterial operons directly in plastidshave met with limited success, operons are usually reengineered to optimize them for efficient ex-pression from the chloroplast genome. Common adaptations include replacements of UTRs andintercistronic spacers, codon usage adaptation, and incorporation of intercistronic processing ele-ments (190) (see above). Two general strategies for building plastid operons have proven successful:extension of endogenous plastid operons by additional genes (25, 78) and construction of fullysynthetic operons by using rational design principles (109). An operon extension strategy was ap-plied to optimize the production of the renewable and biodegradable plastic polyhydroxybutyratein tobacco chloroplasts by coexpression of three bacterial enzymes: β-ketothiolase, acetoacetyl-CoA reductase, and polyhydroxybutyrate synthase (25). Principles of synthetic operon design wereworked out using the vitamin E (tocopherol) biosynthetic pathway as an example and the threepathway enzymes homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyl-transferase (109). Although in both these cases only three transgenes were combined in an operon,there is every reason to believe that much larger operons can be constructed according to similarprinciples.
Molecular Farming
Molecular farming represents a growing area of biotechnology that aims to harness the hugepotential of plants as inexpensive factories for the large-scale production of recombinant pharma-ceutical proteins and industrial enzymes. Motivated by the appealing concept of edible vaccinesand in view of the very high foreign protein accumulation levels attainable in transgenic chloro-plasts, much of the initial work on molecular farming in plastids focused on the expression ofantigens for subunit vaccines (40, 61, 67, 123, 175, 189). Indeed, a number of viral and bacterialantigens could be expressed to high levels and proved to be immunogenic when tested in animalmodels using different immunization routes, including, in some cases, oral immunization (8, 35,42, 71, 103). However, despite many promising expression studies and encouraging results fromanimal tests, no chloroplast-produced vaccine has entered the clinic yet.
More recently, several other therapeutic proteins were successfully expressed in transgenicplastids. These include, for example, antibody fragments for passive immunization and/or diag-nostics (102), a blood coagulation factor potentially applicable in hemophilia treatment (182), andseveral endolysins (132, 133). The latter are lytic proteins encoded in the genomes of bacterio-phages that infect and eventually kill pathogenic bacteria. Endolysins are necessary and sufficientto induce the lysis of a bacterial cell and, therefore, hold great promise as future next-generationantibiotics (24).
Many of the efforts to produce industrial proteins in plastids have focused on biofuel enzymes.The surging interest in biomass as a renewable energy source has created a great demand forlarge quantities of cheap enzymes that catalyze the efficient degradation of lignocellulosic mat-ter into fermentable sugars. Representatives of almost all known classes of enzymes involved incell wall degradation have been tested in transplastomic expression studies, including variousendo- and exocellulases, glucosidases, xylanases, pectate lyases, and cutinases (90, 137, 180, 181,187). Many of these enzymes could be expressed to high levels, including some enzymes from
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thermophilic biomass-degrading microorganisms. At the industrial scale, thermophilic enzymesoffer the advantage of allowing enzymatic processing of plant biomass at elevated temperatures,thus protecting the released sugars from unwanted consumption by contaminating microbes.
From the many (successful and unsuccessful) attempts to express pharmaceutical and industrialproteins in plastids, some important lessons have been learned. With very few exceptions (184),there is little evidence for problems with RNA accumulation being causally responsible for thefailure to express a foreign protein to reasonably high levels. Instead, in most cases, proteinstability appears to be the factor that limits transgene expression (12, 13, 45, 57). Unfortunately,very little is known about the determinants of protein (in)stability in plastids (7, 45), makingit currently impossible to predict the expression potential of transgenes in plastids. If a proteinturns out to be unstable in the chloroplast stroma, a possible alternative would be targeting it tothe thylakoid lumen, where a different set of proteases resides (10, 102, 174). In addition, carefulevaluation of the physicochemical properties of the protein to be expressed is highly recommended.Proteins harboring hydrophobic domains that tend to associate with plastid membranes often leadto problems with transgene expression and cause deleterious phenotypic effects (by interferingwith thylakoid biogenesis or function) (76). Concerning posttranslational protein modifications,it seems clear that disulfide bonds are usually faithfully formed in chloroplasts (10, 158). However,for recombinant proteins requiring other posttranslational modifications (e.g., glycosylation orspecific phosphorylation patterns), the chloroplast may not be the best site of production.
Finally, there is a striking difference between seed plants and Chlamydomonas in the efficiencyof transgene expression and the attainable expression levels: As mentioned above, for reasons thatare not entirely clear, the protein accumulation levels achieved in Chlamydomonas chloroplasts areoften much lower than those obtained in seed plant plastids. Although recently some progresshas been made with the production of pharmaceutical proteins in Chlamydomonas chloroplasts,the expression levels reached so far do not nearly approach those regularly obtained in seed plantchloroplasts. Moreover, maximum expression rates seem to require knockout of photosynthesis,thus losing one of the greatest attractions of using plant cells as production hosts of recombinantproteins (117, 141).
SUMMARY AND OUTLOOK
Over the past two decades, chloroplast transformation technology has become a mainstay ofmolecular and genetic research on plastids. In addition, numerous proof-of-concept applications inmetabolic engineering, molecular farming, and resistance engineering have impressively demon-strated the great potential of plastid genome engineering in biotechnology. However, althoughexciting strides have been made in developing new tools for plastid genome engineering and effi-cient transgene expression, progress in expanding the species range of the technology has remainedslow. The vast majority of transplastomic research is still done with the two best-established modelsystems: Chlamydomonas and tobacco. From many fruitless efforts made in the past, the communityhas come to realize that developing workable plastid transformation protocols for new species rep-resents a daunting task and requires long-term investments in tedious optimization work directedtoward the improvement of transformation protocols, tissue culture procedures, and selectionconditions. Both the academic and the industrial sectors seem to largely shy away from makingthese investments.
Unfortunately, in the past, the field has also suffered from the publication of several misleadingreports that made exaggerated claims that turned out to be irreproducible. These cases includedfalse claims about transformation protocols for new species and alternative marker genes forselection of transplastomic cells. Some but not all of these faulty papers have been retracted.
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Therefore, any newcomer to the field is advised to study the literature very carefully and, incase of doubt, consult with two or three recognized experts before investing in laborious andtime-consuming transformation projects that use nonstandard markers or species.
Despite many promising proof-of-concept applications, commercial products from transgenicplastids have not yet entered the market, and presently no transplastomic plants are grown com-mercially in the field or in greenhouses. Nonetheless, the unique attractions of transplastomictechnology make a persuasive case for continued investments in technology development. Inaddition to protocols for major crop species (especially cereals), improved tools for inducibleexpression and repression of plastid (trans)genes as well as tissue-specific and developmentallyregulated expression systems rank high on the wish list of researchers in the field. The emerg-ing field of plant synthetic biology will also benefit greatly from the continued expansion of thetoolbox for plastid genome engineering (149). Because of its small genome size and the uniqueprecision with which the genome can be manipulated, the chloroplast lends itself to the explorationof synthetic biology applications, including the design of synthetic genomes, the large-scale engi-neering of novel metabolic pathways into plants, and the radical redesign of metabolic networks,the photosynthetic apparatus, and perhaps even the entire genetic system of the plastid.
SUMMARY POINTS
1. Stable transformation of the plastid genome is routinely possible in the green alga Chlamy-domonas reinhardtii and a few species of seed plants, but still challenging or not yet possiblein many model species and crops.
2. Integration of foreign DNA into the plastid genome occurs exclusively by homologousrecombination.
3. Particle gun–mediated (biolistic) transformation is the most efficient method for intro-ducing DNA into the plastid compartment.
4. A large toolbox for plastid genome engineering is available, including tools for cotrans-formation, selectable marker gene removal, efficient transgene expression in nongreentissues, and multigene engineering with synthetic operons.
5. Transgenic plastids can be horizontally transferred between related species.
6. Chloroplast transformation allows the study of all steps in plastid gene expression in vivoand the functional analysis of plastid genes by reverse genetics.
7. High transgene expression levels, transgene stacking in operons, and increased transgenecontainment (due to maternal plastid inheritance in most species) make plastid genometransformation a highly attractive technology in metabolic pathway engineering, resis-tance engineering, and molecular farming.
DISCLOSURE STATEMENT
The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
I thank Claudia Hasse for photography and Dr. Stephanie Ruf (both from the Max-Planck-Institutfur Molekulare Pflanzenphysiologie) for discussion, help with artwork, and critical reading of the
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manuscript. I apologize to colleagues whose work could not be discussed because of space con-straints. Work on plastid transformation in my laboratory is supported by grants from the Euro-pean Union (EU-FP7 DISCO 613513 and COST Actions FA0804 and FA1006), the DeutscheForschungsgemeinschaft (FOR 2092 and BO 1482/17-1), the Human Frontiers Science Program(RGP0005/2013), and the Max Planck Society.
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