Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century

DNA-containing cellular compartments in plant cells are the nucleus, plastids, and mitochondria. The nuclear genome, encoding approximately 29,000 to 32,000 genes, is the most common target for biotechnological applications. The number of genes encoded by the plastid and mitochondrial genomes is much smaller, approximately 120 and approximately 60, respectively. The number of processes that may be modified by engineering the organellar genomes is much higher than the number of genes would suggest. That is because approximately 10% of the nuclear gene products are targeted to plastids and approximately 10% to mitochondria (Leister, 2003), making these nucleus-encoded processes amenable to manipulation by plastome engineering. Traits that may be engineered by plastid genome manipulation are not restricted by the plant’s gene content, because incorporating new genes or gene clusters from heterologous sources may expand the plastid’s biosynthetic repertoire beyond what is provided by nature. Engineering of the plastid genomes (plastomes; plastid DNA or ptDNA) was first accomplished in Chlamydomonas reinhardtii, a unicellular green alga (Boynton et al., 1988), followed by plastid transformation in tobacco (Nicotiana tabacum), a flowering plant species (Svab et al., 1990). Since 1988, plastid transformation has been expanded to a diverse group of species (see below). However, commercial applications are lagging behind, and currently no crops are grown commercially utilizing this technology. This review focuses on the principles of plastome engineering and highlights recent developments. Most examples will be described in tobacco, which is the model species of plastid engineering. Additional information on the biotechnological applications of plastid transformation can be found in recent reviews (Bock, 2007; Daniell et al., 2009; Cardi et al., 2010). The target of the transforming DNA, the plastid genome, is highly polyploid. The number of plastids per cell and the number of ptDNA copies per plastid is dependent on the species and the cell type. For example, an Arabidopsis (Arabidopsis thaliana) leaf mesophyll cell contains about 120 chloroplasts, and these harbor approximately 1,000 to 1,700 copies of the approximately 154-kb plastid genome (Zoschke et al., 2007). Tobacco leaf cells contain a comparable number (approximately 100) of chloroplasts harboring approximately 10,000 copies of the approximately 156-kb ptDNA (Shaver et al., 2006). The ptDNA in chloroplasts is localized to membranes in clusters of approximately 10 ptDNA copies, referred to as nucleoids (Fig. 1A; Maliga, 2004). Obtaining plants with a genetically uniform population of transformed plastid genomes (transplastomes or T-ptDNA) is still the bottleneck that hinders rapid extension of the technology to new crops. Figure 1 depicts the stages of plastid sorting that ultimately yield genetically stable transplastomic plants and are discussed below. Sorting of T-ptDNA at the organelle and cellular levels yields homoplastomic plants. A, Replication and sorting of T-ptDNA at the organelle level yields homoplastomic organelles. Sorting is facilitated by the conversion of chloroplasts (CHL) to proplastids (PP), which contain only one to two nucleoids instead of 10. Wild-type ptDNA and T-ptDNA (blue circles and red circles, respectively) are anchored to membranes by proteins (black dots) in nucleoids (N). Sorting of ptDNA and T-ptDNA in heteroplastomic nucleoids (#1) yields nucleoids with only T-ptDNA (#1a) and wild-type ptDNA (#1b). For details, see Maliga (2004). B, Division and sorting of plastids yields genetically stable transplastomic plants. Sorting is accelerated by reduction from approximately 100 chloroplasts in leaf cells to approximately 10 to 14 proplastids in meristematic cells. In the cells, the nucleus (Nu) is also marked. C, The plastid genotype of long-term stem cells in the three layers (L1, L2, L3) of the shoot apex determines the plastid genotype in leaves. PZ and CZ are the peripheral and central zones, respectively. On the left is a shoot apex with T-ptDNA in all three layers and a homoplastomic plant carrying only T-ptDNA encoding the aurea spectinomycin resistance (aadA) gene. The variegated plant in the middle has cells with wild-type ptDNA and T-ptDNA in its shoot apex. The regenerated plant on the right has only wild-type ptDNA. C is modified from Lutz and Maliga (2008); the plants were described by Tungsuchat-Huang et al. (2011). When the transforming DNA is introduced on the surface of microscopic particles (Fig. 1, A and B), only one or a few chloroplasts in a leaf cell may be damaged by the impact, and only a few of the approximately 100 ptDNA copies incorporate the transforming DNA. However, the transforming DNA carries antibiotic-detoxifying genes conferring a selective advantage to plastids that carry the T-ptDNA. Because the selective agent can most conveniently be administered in the tissue culture environment, selective enrichment for the T-ptDNA is carried out in tissue culture cells. The tissue culture medium triggers cell division, yielding meristematic cells with 10 to 14 proplastids, each of which carries only one or two nucleoids. Reduction of plastid number from 100 to 10 to 14 greatly accelerates plastid sorting during cell division, during which plastids carrying the T-ptDNA are dividing at a faster rate (Fig. 1B). Plastids carrying only the wild-type ptDNA are ultimately lost by dilution during cell division. Depicted in Figure 1 is a selection for an aurea construct that confers a pale pigment phenotype to plants (Lutz and Maliga, 2008; Tungsuchat-Huang et al., 2011). In tobacco, selection for T-ptDNA is carried out on a shoot induction medium. Cells carrying T-ptDNA are relieved from inhibition by the toxic selective agent and will regenerate shoots on the selective medium (Fig. 2). Cells in the shoot derive from three developmental layers, each of which is the progeny of two to three slowly dividing long-term stem cells (Fig. 1C). Genetic uniformity of a regenerated plant is ensured when each of the long-term stem cells in the three developmental layers carries the same T-ptDNA. Shoots emerging from a bombarded leaf are chimeric, consisting of transplastomic and wild-type sectors. The transplastomic sectors, often very small, can be visualized by the expression of aurea transgenes that confer a golden-yellow phenotype to leaves (Fig. 1C, bottom middle). The pigment phenotype is due to posttranscriptional interference with the plastid clpP1 (ATP-dependent protease proteolytic subunit) gene expression by the aurea aadAau (aminoglycoside 3′′-adenylyltransferase, aurea) transgene. Wild-type sectors in the shoot are present because transplastomic sectors protect wild-type cells against antibiotics in culture. Cross-protection enables wild-type shoots to form on a selective medium even after two cycles of shoot regeneration. Genetically stable transplastomic plants can be obtained by collecting seeds from plants with a uniform phenotype after two cycles of plant regeneration or from branches that carry transplastomic sectors in the second leaf layer, the source of germline cells (Lutz and Maliga, 2008; Tungsuchat-Huang et al., 2011). Because heteroplastomic cells are rare even in variegated plants and are localized at the edge of sectors, the aurea and green colors typically identify homoplastomic transgenic and wild-type sectors, respectively. Plants in Figure 1C were transformed with an aurea aadA gene that is selectable in culture and gives a visual phenotype in leaves (Tungsuchat-Huang et al., 2011). In the absence of a visual marker, DNA gel-blot analysis is employed to identify plants with a uniform population of T-ptDNA molecules (Maliga, 2004). Transplastomic clones are identified as green shoots in bombarded tobacco leaf culture on spectinomycin medium. The aurea aadAau gene (Tungsuchat-Huang et al., 2011) enables greening and shoot regeneration in the culture shown here and causes intense golden-yellow leaf pigmentation in plants (Fig. 1C). Incorporation of foreign DNA is based on homologous recombination between the targeting region of the vector and the ptDNA (Fig. 3). The transformation vectors are Escherichia coli plasmids that do not replicate in plastids; thus, the marker gene encoded in the vector will be stably expressed only if incorporated in the plastid genome by homologous recombination. The transforming DNA is introduced on the surface of microscopic (0.4–1.0 μm) gold or tungsten particles or by polyethylene glycol treatment. Biolistic DNA delivery is used when the targets are plastids in intact tissue; polyethylene glycol treatment is used for DNA introduction into protoplasts (Dix and Kavanagh, 1995). The most commonly used selective marker gene is aadA, encoding spectinomycin resistance (Svab and Maliga, 1993). Kanamycin (Carrer et al., 1993), chloramphenicol (Li et al., 2011), and the amino acid analogs 4-methylindole and 7-methyl-dl-Trp (Barone et al., 2009) have also been successfully employed as selective agents. Plastid genome manipulation is based on homologous recombination between ptDNA and the targeting regions in the vector. A, Replacement of the tobacco rbcL gene (T-rbcL) with the sunflower homolog (S-rbcL). The sunflower S-rbcL is incorporated in the tobacco ptDNA only if recombination is via the atpB and accD genes (dotted lines). Recombination adjacent to aadA (arrows) confers spectinomycin resistance, but the tobacco T-rbcL is retained. Out of six transplastomic lines, three carried aadA only, two incorporated S-rbcL, and one had recombination within rbcL (Kanevski et al., 1999). B, Insertion of the lux operon in the trnI/trnA intergenic region. Note that the lux operon is transcribed from the aadA promoter and the gene cluster has only a single 3′ UTR (Krichevsky et al., 2010). The types of plastid genome manipulations include knockout of plastid genes to probe function, replacement of plastid genes with mutant forms, and insertion of transgenes to confer novel functions. Replacement of the tobacco rbcL plastid gene (T-rbcL), encoding the Rubisco large subunit, with the sunflower (Helianthus annuus) large subunit (S-rbcL) is shown in Figure 3A (Sharwood et al., 2008). The example shown in Figure 3B is insertion of aadA and six genes (approximately 6.5 kb) of the luciferase (lux) operon in the plastid genome in the trnI-trnA intergenic region (Krichevsky et al., 2010). Thus far, this is the highest number of genes inserted in the ptDNA. In both cases, the genes of interest (S-rbcL and lux operon) were incorporated in the plastid genome by homologous recombination via the homologous flanking (“targeting”) sequences (dashed lines in Fig. 3). The recovery of T-ptDNA was subsequently facilitated by selection for the linked spectinomycin resistance (aadA) marker gene. Once a uniform population of engineered T-ptDNA has been obtained, the marker genes are no longer necessary to maintain the T-ptDNA. Excision of the marker gene enables multistep transformation with the same marker gene. The metabolic burden from high-level expression of the marker gene and opposition to antibiotic resistance markers in field crops are additional reasons that make the removal of marker genes desirable. Protocols for marker gene removal employ the Cre and Int phage site-specific recombinases that excise the marker genes via flanking recombinase target sites (Fig. 4; Lutz and Maliga, 2007). Alternatively, the marker gene may be removed by recombination via flanking direct repeats (Fig. 5; Kode et al., 2006). Excision of marker genes by site-specific recombinase enzymes. A, Marker genes in the plastid transformation vectors are flanked by loxP or attP/attB sequences (triangles) that are the targets for site-specific recombinases. B, The marker genes are efficiently removed when a gene encoding a plastid-targeted Cre or Int recombinase is introduced into the nucleus by transformation or pollination (Lutz and Maliga, 2007). T1-ptDNA and T2-ptDNA refer to the marker-containing and marker-free transplastomes. Marker-free plastids by repeat-mediated deletion of the marker gene. In the transplastome (T-ptDNA), the aadA marker gene, expressed in the P2/T2 cassette, disrupts the hppd herbicide tolerance gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme in the tocopherol biosynthetic pathway. The hppd coding region is flanked by the P1/T1 cassette but is not expressed due to disruption by aadA. Note the 403-nucleotide duplicated segment flanking the aadA. of aadA via the 403-nucleotide repeats a hppd gene the expression of herbicide resistance in et al., 2007; for see Kode et al., 2006). of the technology of plastid transformation to new crops has been than nuclear gene are of in for plastid transformation have been described only in tobacco (Svab and Maliga, 1993), et al., et al., et al., 2011), et al., et al., and et al., 2007). as a group to be the most of transgenes is facilitated by the of expression into which coding regions can be inserted (Fig. are in plastid transformation vectors that also as coli that vectors can be obtained in one The regions are provided by a cassette, which a promoter and sequences The sequences may be the region or the region that the UTR and the coding The of the UTR is to the and to of the of the is facilitated by by a of the found of the much A number of as have no and proteins are to in these of of the is dependent on et al., 2007). The 3′ region or cassette the 3′ which typically a The 3′ region is for et al., in for A, are of transgenes transcribed from a and a and sites may be present in the same cassette the and each of which the in are B, DNA of the promoter with the atpB UTR et al., and and Maliga, Note that expression in the cassette yields a with 14 amino from the plastid atpB gene and two amino encoded in the the an In are shown the promoter and promoter Most biotechnological applications the promoter of the plastid is the plastid but its are not is with the UTR or of plastid or of a a cassette enables expression of the with its and C because the expression are linked with the coding region via sites the and (Fig. a the of interest with an is the only that yields high-level are expression of the enzyme from the coding region et al., or the et al., 2008; Fig. The engineered plastid UTR is typically a and mutant form of the the of the UTR is to the of homologous recombination the UTR and are introduced to For example, the highly expressed cassette approximately 10% of contains only of the rbcL and carries a to an and Maliga, The of the was shown by as much as on the of (Maliga, The most sequences derive from the coli phage and Maliga, the et al., and the plastid rbcL gene in its engineered form and Maliga, The highest level of expression in chloroplasts on is than of of a highly stable antibiotic in a cassette et al., see below). of at in cases, is dependent on of into of is a encoding an expression may be inserted to et al., 2007). The of the insertion in the plastome may have a on the level of a in the region of the ptDNA the number of copies per genome, as with in Insertion of transgenes between genes of a transcribed operon will the level of typically yielding higher levels et al., no transplastomic crops are grown However, of in tobacco for applications. is by the of the technology in field crops as and or only recent of the technology in The first example the of the transplastomic technology from the expression of the in chloroplasts et al., 1995). The coding region of the genes, when expressed in the plant nucleus, was that the genes are as with plant nuclear genes, which to the that expression was due to of proteins from a coding region with an from to to of cellular However, when the coding region was expressed in chloroplasts, the was stable and the to to of the cellular which was at the In the proteins have been expressed in chloroplasts with a high-level of the from the coding The levels obtained were than 10% in tobacco et al., and et al., and when two of the one of which a the to of the et al., resistance is one of the most common commercial transgenic in commercial crops are encoded in the nucleus, for of the herbicide resistance by to the same of have been obtained by expression of transgenes in include resistance to et al., and Lutz et al., et al., and et al., 2008). these plastid transgenes are incorporated in commercial plastid an et al., 2007; and Maliga, 2007). of transgenes in the plastid genome to confer herbicide resistance is an example for of a nucleus-encoded a nucleus-encoded metabolic that is to an by plastome engineering. the of by engineering of the is the of plant the technology is very has been The only is the large subunit of which is encoded in the of has to the of by Rubisco engineering to a new green et al., 2011). engineering for the yield and of and can an and source of for processes to the by plastid transformation have been et al., 2007; et al., 2008; and 2009; Fig. are and an of the The referred to as is of because the for A, a to all A is a the A of crops an of and engineering advantage of the of a plastid transformation for a plant that of in during the A in transplastomic was the of red can be into in a by (Fig. genes from source and were to plastid expression and as transgenes in transplastomic plants et al., 2007; and of was most with a plant gene from with A levels as much as 1 of the wild-type have than A and 2009; Fig. expression of the from the plastid genome not only in conversion of to also to a in content, a additional in the of the This that the an in at in determines the the in the engineering of the in transplastomic plants. A, of the biosynthetic pathway. the in is in and is in that have been used for plastid genome engineering are in of the not in higher plants are shown in The of the are by B, of from transplastomic plants a from from a wild-type plant two and a transplastomic two were at stages and from the and from the The of the transplastomic from the conversion of the red into the A The A levels 1 wild-type had than A This is modified from and The of novel was in a in tobacco et al., 2008). The is a that is used as a and in is for the of the and for to of the also has applications in the and due to its much higher with most is not by higher plants but in and as and two and (Fig. of genes for the two from a of the in tobacco chloroplasts in to than of the of the that chloroplasts can of novel to expression in this also in a in the transplastomic tobacco plants et al., 2008). Because of to chloroplasts were a for the of acid the expression of three In the most the genes were expressed from the promoter et al., The was transcribed by a plastid-targeted The first gene, was from the and were from the intergenic of in chloroplasts that is between the plastid and to the proteins from the with was by the plastid-targeted from an of plants the expression of a luciferase consisting of six genes et al., 2010). the first gene of the lux was expressed from the plastid cassette, and the of the lux operon genes were expressed from of by the to the plastid’s to the expression of plastids, making amenable to engineering by plastid of the plastid accD gene, encoding the subunit of leaf et al., of a gene in tobacco chloroplasts from the species or the in acid and an in level both in leaves and seeds et al., 2008). the interest in by plastid engineering will a of the most of transplastomic plants is to foreign proteins (Maliga, 2007; Daniell et al., This at in due to the number of chloroplasts per the large of the cell by the and the number of the plastid genome, with or even of copies present in a single The enzyme Rubisco a in for the of the large subunit is encoded in the plastid genome, and in the Rubisco enzyme can to than to of the in leaves et al., 2011). yields of are to the of proteins in an of commonly referred to as for example, and proteins can be from the plant in the of expressed in plants and administered In both cases, expression levels are of The of make by the of the for and in with expression levels are for the of because of the much higher of the than by into the the of in expression of a large number of has been in chloroplasts recent see and Cardi et al., 2010). levels have been from to as much as of the et al., et al., 2008). However, far, very few of the have successfully been in et al., et al., 2010). all have been to and the of with transplastomic plants still its in large and ultimately in expression levels have been obtained with antibiotics from phage proteins proteins efficiently the cell of for this have as antibiotics to that have resistance to most antibiotics that are currently in The may be an for the of these antibiotics because chloroplasts do not have a cell be to large of these proteins and chloroplasts a very of as The is that phage proteins have levels of resistance to by When expressed from the tobacco plastid genome, against the agent of out to be stable et al., levels of to than of the of the plant et al., the were highly and efficiently the target The expression levels also make transplastomic plants an for as enzymes. the interest in and enzyme that are to into have recent have that the can very levels of these and et al., 2007; et al., et al., 2011), can mutant and 2011). For expressed to levels in to is often and can be transplastomic plants cell can a source of for the of However, in the conversion of into to be in to make an and the for of the to be This will for and removal or of the A number of recent have the in of the unicellular green alga Chlamydomonas et al., 2011). in is than of higher plants in these are of an in with the if one that proteins are products and are in in by a on in and has been with proteins from the genome of In the of the Chlamydomonas to foreign proteins to be much than that of higher plants. in flowering of regions is et al., of expression are to of cellular et al., 10% and expression levels were also obtained from the expression in knockout et al., 2007; et al., expression in the knockout plastids may be due to of expression by the of for these by the Incorporation of at an and expression from heterologous and of the at a level et al., 2007). on the of Chlamydomonas in which of expression have been removed by nuclear et al., 2009; et al., 2011). Plastid engineering is currently in three and flowering plants. Plastid transformation was first in a However, with Chlamydomonas was when of the of the expression of proteins at commercially levels et al., Chlamydomonas is to a expression for the same the is on for new species that may be for Plastid transformation in the and and et al., is recent and has not been for the expression of However, by targeted nuclear gene were with and the is grown in a tissue culture for were to be to currently used cell lines in with and 2007). a is expression will the of the A in flowering plants will be the of the technology in a for applications. in new crops will on the of a that is for cycles of plant regeneration and a selectable marker gene, as discussed as and applications are in of and flowering will be the source of enzymes. The tobacco plastid expression is to this However, is not for the of due to its the the as a for the expression of et al., 2007). is a for high-level gene expression that and the can be and the of proteins to of are et al., and expression et al., currently still the that is for applications.