JOURNAL OF

GENETICS AND GENOMICS

J. Genet. Genomics 36 (2009) 387398 www.jgenetgenomics.org

Advances in chloroplast engineering

Huan-Huan Wang a, b, Wei-Bo Yin a, Zan-Min Hu a, *
a

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China Received for publication 12 January 2009; revised 30 April 2009; accepted 4 May 2009

Abstract The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the worlds food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus: high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.
Keywords: chloroplast (plastid) transformation; homologous recombination; protein expression; marker excision

Introduction
The chloroplast is one of organelles known as plastids in plant cells and eukaryotic algae. It is the site of photosynthesis, which provides the primary source of the worlds food productivity. Other important activities that occur in plastids include evolution of oxygen, sequestration of carbon, production of starch, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism (Verma and Daniell, 2007). Plastids are plant cellular organelles with a ~120150 kb genome present in 1,00010,000 copies per cell (Bendich, 1987), and maternally inherited in most angiosperm plant species (Hagemann, 2004). Since the accomplishment of genetic transformation of chloroplasts two decades ago (Boynton et al., 1988; Svab et al., 1990), chloroplast
* Corresponding author. Tel & Fax: +86-10-6480 7626. E-mail address: zmhu@genetics.ac.cn DOI: 10.1016/S1673-8527(08)60128-9

transformation has become an attractive alternative to nuclear gene transformation due to its advantages: high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission (Maliga, 2002; Bock, 2007; Kittiwongwattana et al., 2007). In this paper, we review and highlight recent studies of chloroplast engineering.

The procedures of chloroplast transformation The transformation method development

Chloroplast transformation was generally achieved by the biolistic process, with which the Escherichia coli plasmids containing a marker gene and the gene of interest were introduced into chloroplasts or plastids. The foreign genes were inserted into plasmid DNA by homologous

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recombination via the flanking sequences at the insertion site. Agrobacterium mediated method was firstly used in plastid transformation (Block et al., 1985). PEG-mediated transformation method was also applied in the early days (Sporlein et al., 1991; Golds et al., 1993; ONeillt et al., 1993) and a recent study (Craig et al., 2008). A review of the method (Koop et al., 1996) and a step-by-step protocol of plastid transformation by PEG treatment are available (Koop and Kofer, 1995). Since the first successful chloroplast transformation in Chlamydomonas reinhardttii (a unicellular algae with a single chloroplast) by bombardment method (Boynton et al., 1988), this method has become a favorable means for chloroplast or plastid transformation due to its higher transformation efficiency and simple operation. Most of successful chloroplast transformations were achieved by this method. Protocols of plastid transformation by bombardment method are available

(Koop and Kofer, 1995; Lutz et al., 2006b). After the first chloroplast transformation in Chlamydomonas Reinhardttii (Boynton et al., 1988), the first stable plastid transformation was established soon in higher plants, Nicotiana tabacum (Daniell et al., 1990; Svab et al., 1990). Up to date, plastid transformation has extended to many other higher plants, such as Arabidopsis (Sikdar et al., 1998), rape (Hou et al., 2003), Lesquerella (Skarjinskaia et al., 2003), rice (Lee et al., 2006), potato (Sidorov et al., 1999), lettuce (Lelivelt et al., 2005), soybean (Dufourmantel et al., 2004), cotton (Kumar et al., 2004a), carrot (Kumar et al., 2004b), tomato (Ruf et al., 2001) and poplar (Okumura et al., 2006) (see review written by Verma and Daniell, 2007, and Table 1). However, plastid transformation is routine only in tobacco and the efficiency of transformation is much higher in tobacco than in other plants (Maliga, 2004).

Table 1 Chloroplast transformation methods and selection conditions for different plant species Plant spieces Chlamydomonas reinhardtii Nicotiana tabacum (tobacco) Tobacco Tobacco Tobacco Tobacco Tobacco Tobacco Arabidopsis thaliana Solanum tuberosum (potato) Rice Lycopersicon esculentum (tomato) Brassica napus (oilseed rape) Lesquerella fendleri Daucus carota (carrot) Gossypium hirsutum (Cotton) Glycine max (Soybean) Petunia hybrida Lactuca sativa (lettuce) Lettuce Brassica oleracea (cauliflower) Populus alba (poplar) Brassica oleracea L. var. capitata L. (cabbage) Methods Bombardment Bombardment PEG Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment Bombardment PEG Bombardment PEG & Bombardment Bombardment Bombardment Selection photosynthetic competence Spectinomycin Spectinomycin Kanamycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Kanamycin Spectinomycin Spectinomycin & streptomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin Spectinomycin & streptomycin Expressed genes atpB rrn16 rrn16 nptII uidA Human somatotropin(hST) Bt Bar & aadA aadA aadA & gfp aadA & gfp aadA aadA & cry1Aa10 aadA & gfp dehydrogenase (badh) aphA-6 aadA aadA & gusA gfp gfp gus & aadA gfp aadA & uidA Literature cited Boynton et al., 1988 Svab et al., 1990 Golds et al., 1993 Carrer et al., 1993 Staub and Maliga, 1995 Staub et al., 2000 Cosa et al., 2001 Lutz et al., 2006a Sikdar et al., 1998 Sidorov et al., 1999 Lee et al., 2006 Ruf et al., 2001 Hou et al., 2003 Skarjinskaia et al., 2003 Kumar et al., 2004b Kumar et al., 2004a Dufourmantel et al., 2004 Zubko et al., 2004 Lelivelt et al., 2005 Kanamoto et al., 2006 Nugent et al., 2006 Okumura et al., 2006 Liu et al., 2007

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Vector design Selection marker genes Since ptDNA (plastid DNA) is present in many copies, selectable marker genes are critically important to achieve uniform transformation of all genome copies during an enrichment process that involves gradual sorting out non-transformed plastids on a selective medium (Maliga, 2004; Kittiwongwattana et al., 2007). The first selection marker gene used in chloroplast transformation was plastid 16S rRNA (rrn16) gene (Svab et al., 1990). Transgenic lines were selected by spectinomycin resistance and the efficiency was low. Subsequently, the aadA gene encoding aminoglycoside 3-adenylyltransferase was used as a selection marker gene (Goldschmidt-Clermont, 1991; Svab and Maliga, 1993). Transformation with aadA gene dramatically improved the recovery of plastid transformants to a rate of, on average, about one transplastomic line in a bombarded leaf sample (Svab and Maliga, 1993). The npt II was also used as a selectable marker for plastid transformation in tobacco, but the transformation efficiency was low, i.e. about one transplastomic line per 25 bombarded samples (Carrer et al., 1993). A dramatic improvement in plastid transformation efficiency was obtained by a highly expressed neo gene. Bombardment of 25 leaves with the vector that carries the new neo gene (pAAK201) yielded 34 kanamycin resistant clones (Kuroda and Maliga, 2001b). The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT), has also been tested as a marker gene, but it was not good enough (Lutz et al., 2001). Another marker gene is the betaine aldehyde dehydrogenase (BADH) gene which confers resistance to betaine aldehyde. Chloroplast transformation efficiency was 25-fold higher with betaine aldehyde (BA) selection than with spectinomycin in tobacco (Daniell et al., 2001b). Transgenic carrot plants expressing BADH could be grown in the presence of high concentrations of NaCl (up to 400 mmol/L) (Kumar et al., 2004b). But there is no additional report about the use of BA selection. Insertion sites Plastid expression vectors possessed left and right flanking sequences each with 12 kb in size from the host plastid genome, which are used for foreign gene insertion into plastid DNA via homologous recombination. The site of insertion in the plastid genome is determined by the choice of ptDNA segment flanking the marker gene and the gene of interest. Insertion of foreign DNA in intergenic regions of the plastid genome had been accomplished at 16

sites (Maliga, 2004). Three of the insertion sites, trnV-3'rps12trnI-trnA and trnfM-trnG, were most commonly used (Maliga, 2004). The trnV-3'rps12 and trnI-trnA sites are located in the 25 kb inverted repeat (IR) region of ptDNA and thus a gene inserted into these sites would be rapidly copied into two copies in the IR region. The trnfM-trnG site is located in the large single copy region of the ptDNA, and the gene inserted between trnfM and trnG should have only one copy per ptDNA. The pPRV series vectors targeting insertions at the trnV-3'rps12 intergenic region are the most commonly used vectors in tobacco. They yield high levels of protein expression (Maliga, 2003) and were endowed with signals for marker gene excision (Lutz et al., 2006b). The first vector targeting insertions in the trnI-trnA intergenic region, pSBL-CTV2, was developed in the Daniell laboratory (Daniell et al., 1998) and was used to express several proteins (Daniell et al., 2005a). Several laboratories have inserted transgenes between the trnI and trnA genes in several plant species (Daniell et al., 1998). These two tRNAs are located between the small (rrn16) and large (rrn23) rRNA subunit genes and the operon is transcribed from promoters upstream of rrn16. The polycistronic rrn operon mRNA is efficiently processed, releasing the transgenic mRNA inserted between the two tRNAs.

Regulation sequences The gene expression level in plastids is predominately determined by promoter and 5-UTR elements (Gruissem and Tonkyn, 1993). Therefore, suitable 5-untranslated regions (5-UTRs) including a ribosomal binding site (RBS) are important elements of plastid expression vectors (Eibl et al., 1999). In order to obtain high-level protein accumulation from expression of the transgene, the first requirement is a strong promoter to ensure high levels of mRNA. Most laboratories used the strong plastid rRNA operon (rrn) promoter (Prrn). Stability of the transgenic mRNA is ensured by the 5-UTR and 3-UTR sequences flanking the transgenes. Protein accumulation from the transgene depends on the 5-UTR inserted upstream of the open reading frame encoding the genes of interest. Protein accumulation from the same (Prrn) promoter may vary as much as 10,000-fold depending on the choice of translation control signals (Eibl et al., 1999; Kuroda and Maliga, 2001a, 2001b; Zou et al., 2003). The most commonly used 5-UTR and 3-UTR is psbA/TpsbA (Zoubenko et al., 1994; Milln et al., 2003; Watson et al., 2004; Daniell et al., 2005b; Kittiwongwattana et al., 2007). More information is available in several reviews (Maliga, 2003; Lutz et al.,

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2007; Verma and Daniell, 2007).

Controlled expression of plastid trans-gene in plants

A major drawback in the engineering of plastid gene expression is the lack of tissue-specific developmentally regulated control mechanisms. There is a clear need to develop tightly controllable systems for transgene expression in the chloroplast genome. It is highly desirable to limit transgene expression to certain tissues, organs and/or developmental stages for following reasons. First, highlevel expression of foreign proteins may have deleterious phenotypic effects (Magee et al., 2004) and impose a significant metabolic burden on the plant. Deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. And if plastid transformation carried out in food crops, inadvertent contamination of the food chain with the plant-produced chemical or pharmaceutical must be prevented. This could be accomplished by making transgene expression dependent on an inducer. To establish such a system, a -glucuronidase (GUS) reporter gene under the control of phage T7 gene 10 promoter was introduced into the plastid genome of plants. GUS expression was dependent on nuclear-encoded plastid targeted T7 RNA polymerase (T7 RNAP) activity. First, a T7 RNAP chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco chloroplast transit-

peptide sequence was introduced into tobacco by nuclear transformation. Then plastid transformation was carried out by using the plastid vector contained a -glucuronidase (GUS) reporter gene under the control of T7 promoter. The crossing between the nuclear transformed plants and plastid transformed plants demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNAP gene from the male parent. GUS expression was dependent on nuclear-encoded plastid targeted T7 RNAP activity (McBride et al., 1994). In order to induce polyester polyhydroxybutyric acid (PHB) synthesis in tobacco in a well-timed manner, a trans-activation system to regulate transcription of the phb operon in plastids was constructed. It was an ethanol-inducible T7 RNAP system (Fig. 1). This system consists of a nuclear-located, ethanol-inducible T7 RNAP which is targeted to plastids harboring the phb operon under control of T7 regulatory elements. Following treatment with 5% ethanol, moderate induction of PHB synthesis was found. Without ethanol induction, development of flowers and fertile seeds was possible. Thus, the main problem of inhibitory transgene expression was solved (Lossl et al., 2005). More recently, a Lac repressor-based IPTG-inducible expression system for plastids has been reported, although transgene repression in the uninduced state was incomplete (Muhlbauer and Koop, 2005). This is a system for external control of plastid gene expression which is based entirely on plastid components and can therefore be established in

Fig. 1. An inducible expression system in plastids. T7 RNAP with a small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. The T7 RNAP was expressed under control of an inducible promoter. Induction by ethanol application leads to import of the T7 RNAP into the plastids. Transcription of plastid transgenes under control of T7 promotor occured and the foreign gene expressed in the chloroplasts (Lossl et al., 2005).

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a single transformation step. It uses modified promoters containing binding sites for the bacterial lac repressor. So chemical induction can be made with intact plants or after harvesting (Muhlbauer and Koop, 2005). This will be one of the major groundbreaking developments in plastid biotechnology.

Application of chloroplast engineering Improvement of plant traits

Several environmental stresses such as disease, drought, insect pests, salinity and freezing, can severely limit plant growth and development. In order to improve the plant traits, many researchers had done a series attempts. Many important agronomic traits have already been engineered via the plastid genome, such as herbicide resistance, insect resistance, and tolerance to drought and salt.

Biotic stresses The insect resistance genes were investigated for high-level expression from the chloroplast genome. cry genes could be expressed extremely well in the plastid genome and there was no requirement to adjust the codon usage nor any need for other sequence manipulation (Kota et al., 1999; Cosa et al., 2001). Consequently, leaves of these transplastomic plants proved highly toxic to herbivorous insect larvae. A recent report that high-level expression (about 10% of total soluble protein) of a cry gene (Cry9Aa2) from the plastid genome resulted in severe growth retardation (Chakrabarti et al., 2006), indicating that, rather than maximizing expression, a suitable expression level is required to obtain plants with good insect protection while minimizing yield penalty. The recent generation of insect-resistant transplastomic soybean plants offers optimism for the transfer of the technology to important (food) crops (Dufourmantel et al., 2005). Plant diseases have affected global crop production. Transgenic chloroplasts conferred resistance to the fungal pathogen Colletotrichum destructive in tobacco (DeGray et al., 2001). The chloroplasts were estimated to express MSI-99 at 21.5% to 43% percent of total soluble protein (TSP). MSI-99 was expressed at high levels to provide 88% (T1) and 96% (T2) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. This data suggests that MSI-99 expressed in tobacco chloroplasts can offer significant protection from both bacterial and fungal pathogens.

Abiotic stresses Chloroplast engineering had been successfully applied for the development of plants with tolerance to salt, drought and low temperature. Previous research has shown that over-expression of enzymes for Glycine betaine GlyBet biosynthesis in transgenic plants improved tolerance to various abiotic stresses (Rhodes and Hanson, 1993). Choline monooxygenase (BvCMO) from beet (Beta vulgaris), the enzyme that catalyzes the conversion of choline into betaine aldehyde, has been recently transferred into the plastid genome of tobacco. Transplastomic plants demonstrated that higher photosynthetic rate and apparent quantum yield of photosynthesis in the presence of 150 mmol/L NaCl. Salt stress caused no significant change on the maximal efficiency of PSII photochemistry (Fv/Fm) in both wild type and transplastomic plants (Zhang et al., 2008). Transplastomic carrot plants expressing BADH could be grown in the presence of high concentrations of NaCl (up to 400 mmol/L), the highest level of salt tolerance reported so far among genetically modified crop plants (Kumar et al., 2004b). Trehalose has been found to accumulate under stress conditions such as freezing, heat, salt, or drought, so that it is thought to play a role in protecting cells against damage caused by these stresses. In contrast to nuclear transgenic plants that exhibited pleiotropic effects even at low levels of TPS1 expression, chloroplast transgenic plants grew normally and accumulated trehalose 25-fold higher (Lee et al., 2003). Chloroplast transgenic plants showed a high degree of drought tolerance by remaining green and healthy in 6 percent PEG, whereas wildtype plants were completely bleached (Lee et al., 2003). The unsaturation level of fatty acids (FA) in plant lipids has several implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. 9 desaturase gene, an important gene in lipid metabolic pathways, was transformed into tobacco chloroplast. The transplastomic plants demonstrated the feasibility of using plastid transformation to engineer lipid component in both vegetative and reproductive tissues for increasing cold tolerance (Craig et al., 2008). The feasibility to use chloroplast genetic engineering for weed control has been explored in several studies that aimed at producing glyphosate-tolerant tobacco plants (Daniell et al., 1998; Lutz et al., 2001). Plastid expression of the bar gene encoding the herbicide-inactivating phosphinothricin acetyltransferase (PAT) enzyme led to high-level enzyme accumulation (up to > 7% of TSP) and conferred field-level tolerance to glufosinate (Lutz et al.,

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2001). These cases demonstrate that transplastomic technology might be particularly useful to develop plants resistant to abiotic and biotic stresses. The plastids engineered to have an adequate expression of resistance genes provide effective plant protection in the field.

(Faye and Daniell, 2006). If glycoproteins could be expressed, and expressed proteins have normal glycosylation sites and functions, engineering chloroplasts would be used in more aspects. Further studies will be needed to study the glycoproteins expression and the mechanism of glycosylation in the chloroplasts.

Production of biopharmaceuticals
A therapeutic protein, human serum albumin (HSA) was firstly expressed in transgenic chloroplasts of tobacco at an expression level up to 11.1% of TSP, which is 500-fold greater than the nuclear expression (Milln et al., 2003). So far, most efforts have been focused on the high-level production of antigens for use as vaccines and their tests for immunological efficacy in animal studies. Cholera toxin B sub-unit (CTB) of Vibrio cholerae, a candidate vaccine antigen, has been expressed in chloroplasts resulting in an accumulation of up to 31.1% of TSP as functional oligomers (Daniell et al., 2001a). An animal vaccine epitope, 2L21 peptide that confers protection to dogs against virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a fusion protein with CTB and with green fluorescent protein (GFP) (Molina et al., 2004). LecA, a potential target for blocking amoebiasis, was expressed in chloroplasts to yield up to 6.3% of TSP or 2.3 mg LecA/g leaf tissue (Chebolu and Daniell, 2007). Recently, chloroplast transformation of the high-biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV-1) p24 antigen (McCabe et al., 2008). Chloroplast system is most suitable for high-level expression and economical production of therapeutic proteins in an environmentally friendly manner. However, the cost for purification of these proteins can be eliminated if they are orally delivered or minimized by the use of novel purification strategies. Oral delivery of therapeutic proteins is emerging as a new alternative for medical treatment and will benefit those who cannot afford the high cost of current treatments. As described above, transgenic chloroplasts have been used for the production of many therapeutic proteins, It suggests that the chloroplast should contain the mechanism that allows correct folding and disulfide bond formation, resulting in fully functional proteins. Despite such rapid progress in the use of this organelle for plant molecular pharming, no glycoprotein has been expressed in transgenic chloroplasts, because N- or O-glycosylation is required for stability and functionality of many proteins

Metabolic pathway engineering

Plastid genome engineering represents an attractive alternative to conventional nuclear transgene expression for metabolic engineering, mainly because of the greatly increased transgene containment and the possibility to stack several transgenes by linking them in operons. The plastid harbors a large number of metabolic pathways and, for this reason, is also commonly referred to as the biosynthetic centre of the plant cell. In view of its outstanding importance and the fact that many of its components are plastid encoded, photosynthesis is an obvious candidate pathway for metabolic engineering. The most complex metabolic pathway to be introduced into the plastid genome so far is that for the synthesis of the bioplastic polyhydroxybutyrate (PHB). Three enzymes of PHB biosynthesis are co-transcribed into the tobacco plastid genome (Nakashita et al., 2001; Lssl et al., 2003). Significant accumulation of PHB in chloroplasts appeared to cause male sterility and severe growth retardation. A recent study has provided evidence that b-ketothiolase expression is responsible for the male sterility of the transplastomic plants (Ruiz and Daniell, 2005). An improved inducible PHB production system placing the operon under the control of a nuclear-encoded ethanol-inducible T7 RNA polymerase was targeted to plastids to control the phb operon in plastids (Lossl et al., 2005). Up to now, the application of plastid transformation to metabolic pathway engineering was restricted to the model species tobacco. To investigate the possibility of engineering a nutritionally important metabolic pathway of non-green plastids, a recent study used plastid transformation in tomato to alter carotenoid biosynthesis towards producing fruits with elevated contents of provitamin A (-carotene), an important antioxidant and essential vitamin for human nutrition (Ruf et al., 2001; Wurbs et al., 2007). This study demonstrated the feasibility of metabolic pathway engineering through plastid transformation in non-green plant organs. This is the first successful example of engineering a nutritionally important biochemical pathway in non-green plastids by transforming the chloroplast genome. It is an encouraging step toward the applica-

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tion of plastid transformation technologies in food crops.

Research on RNA editing

Plastid transformation played an important role in understanding the RNA editing process. RNA editing in the plastids of higher plants involves only C-to-U conversion. RNA editing in plastids of higher plants was discovered in 1991 (Hoch et al., 1991), and since then has been shown in all higher plant species tested so far (Bock, 2000; Tsudzuki et al., 2001). The most complete information is available for the psbL editing site. psbL is a plastid photosynthetic gene, in which the translation initiation codon is created by conversion of an ACG codon to an AUG codon at the mRNA level. In higher plant plastids, most information for the involvement of protein factors is indirect: the existence of species-specific, organelle-specific, and site-specific factors were inferred from genetic experiments. The first evidence for a species-specific editing transfactor was obtained when a spinach editing sequence (psbE) was incorporated in tobacco plastids where it was not edited (Bock et al., 1994), unless the spinach nuclear transfactor was provided by cell fusion (Bock and Koop, 1997). It is assumed that each species has the capacity to edit the sites that it carries, but lacks the capacity for editing the sites that it does not have. But there is one exception, the tomato editing site introduced into the tobacco rps12 gene by plastid transformation is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids (Karcher et al., 2008). Evidence for organelle-specific factors was obtained when an edited Petunia mitochondrial coxII sequence was expressed in tobacco chloroplasts, where none of the seven sites was edited (Sutton et al., 1995). The existence of site-specific factors was inferred from competition between transgenic and native mRNAs. The best studied is the NtrpoB C473, NtpsbL C2, and Ntrps14 C80 cluster in which a group-specific response is attributed to short (23 nucleotide) group-specific sequence elements (Hayes et al., 2006). In plastid transformation, three approaches were used to test mRNA editing: minigenes, translational fusion with a reporter gene, and incorporation of an editing segment in the 3'-untranslated region (3'-UTR). The most simple design was the construction of minigenes, which involved insertion of an editing fragment in a plastid expression

cassette linked to a marker gene (Zoubenko et al., 1994). The second approach, translational fusion with a reporter gene, was used to study the psbL and ndhD editing events that create an AUG translation initiation codon by editing of an ACG codon at the mRNA level ( Chaudhuri and Maliga, 1996). The third approach to test editing was incorporation of editing segments in the 3'-UTR of the aadA marker gene where the editing status of the segment does not affect the expression of the marker gene. In plasmid pRB51, the editing segment can be conveniently cloned in an Xba I-BamH I fragment (Bock et al., 1996). Future studies will focus on understanding the role of RNA editing in plastids. Since both edited and nonedited mRNAs are translated, RNA editing may serve as a regulatory mechanism leading to yielding multiple proteins from the same gene.

The research on safety of chloroplast engineering

Plastids and their genetic information are maternally inherited in most crops and thus are largely excluded from pollen transmission. Expression of the selective marker gene at a high level may be essential during plastid transformation. However, when the homoplastomic plants have been obtained, the marker gene was no longer needed. There is a great concern that the over-use of antibiotics might lead to the development of resistant strains of bacteria. It is desirable to remove the selective marker once it has fulfilled its purpose. Marker-free transplastomic plants can now be obtained though four recently developed protocols: 1) homology-based marker gene excision via directly repeated sequences; 2) excision by phage sitespecific recombinanses; 3) transient cointegration of the marker gene; and 4) the cotransformation-segregation to obtain marker-free plants.

Marker gene excision via directly repeated sequences

The feasibility of the approach was first shown in the chloroplast of the unicellular algae, Chlamydomonas reinhardtii (Fischer et al., 1996). The first report describing the removal of antibiotic resistance genes from higher plant plastid transformants was based on marker excision by loop-out recombination, which occurs because of the presence of short direct repeats on either side of the marker gene (Iamtham and Day, 2000). This system is difficult to control because transformation and marker gene elimination occur simultaneously and the desired deletion deriva-

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tive eventually sorts out in the seed progeny (Maliga, 2002).

Excision by phage site-specific recombinanses

In the Cre-loxP site-specific recombination system, the marker gene and the gene of interest are introduced into the plastid genome in the absence of Cre activity and the marker gene is flanked by two directly oriented lox sites. When elimination of the marker gene is required, a gene encoding a plastid-targeted Cre site-specific recombinase is introduced into the nucleus and, subsequent to its import in plastids, it excises sequences between the loxP sites (Fig. 2). Cre could be introduced by a second transformation mediated by Agrobacterium (Corneille et al., 2001; Hajdukiewicz et al., 2001); it also could be introduced by crossing (Corneille et al., 2001) or by transiently expressing cre from T-DNA by agroinfiltration (Lutz et al., 2006a). The nuclear Cre is subsequently removed by segregation in the seed progeny. In tobacco, introduction of the nuclear cre gene into the nucleus of transplastomic plants by Agrobacterium transformation extends the time needed to obtain marker-free plants by only one month. In an ideal case, it takes about six months to obtain a marker-free transplastomic tobacco plant that expresses a

novel recombinant protein (Maliga, 2003). Another site-specific recombinase that has been tested for marker gene excision is Int, the phiC31 phage sitespecific recombinase. To facilitate excision of the aadA marker gene, it was flanked with directly oriented nonidentical phage attP (215 bp) and bacterial attB (54 bp) attachment sites. Efficient excision of the marker gene was shown after transformation of the nucleus with an Int gene encoding a plastid-targeted Int enzyme (Kittiwongwattana et al., 2007) (Fig. 2). Both the Cre-lox and Int-att site-specific recombination systems are efficient, although Int appears to be the better choice since the ptDNA contains pseudo-lox sites recognized by the Cre but no sequences recognized by the Int (Corneille et al., 2003; Lutz et al., 2004; Kittiwongwattana et al., 2007).

Transient cointegration of the marker gene

Placing the marker gene (aphA-6) outside the targeting region enables selection for a cointegrate structure that forms by recombination via only one of the targeting sequences. The recipient plant is white because the plastid rpoA RNA polymerase subunit gene has been replaced with a spectinomycin resistance (aadA) gene. Introduction of vector DNA by the biolistic process is followed by

Fig. 2. Marker gene excision by the phage Cre or Int site-specific recombinases. Chloroplast-targeted and site-specific recombinase gene (cre or Int) was introduced into the plant nucleus. The expressed recombinase (Cre or Int) excised the marker gene from TP1-ptDNA after importing into chloroplasts. Excision of the marker gene by the recombinases via the target sites (black parts) yielded marker-free TP2-ptDNA carrying only the foreign gene and one recombinant copy of the recombinase recognition sequence (Corneille et al., 2001; Kittiwongwattana et al., 2007; Lutz and Maliga, 2007).

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recombination via the left targeting sequence that yields an unstable cointegrate structure on selective kanamycin medium. When selection for antibiotic resistance is stopped, the second recombination event can take place and the marker gene is lost. If the gene of interest is linked to a missing photosynthetic gene (petA, rpoA), and the recipient lacks this particular gene, marker-free transplastomic plants can be directly recognized by reconstitution of the full gene set restoring green pigmentation (Klaus et al., 2004). This method uses the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. So marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing (Klaus et al., 2004).

site-specific recombinases tested for nuclear gene engineering might be usefully explored for application in plastids (Keravala et al., 2006; Thomson and Ow, 2006). These reports showed that efficient removal of selectable markers from chloroplast genomes is feasible.

Conclusions and future directions

To date, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as to produce industrially valuable biomaterials and therapeutic proteins. The plastid transformation has a high-level of transgene expression. The high expression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as a bioreactor. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. The bacterial genes, and hopefully human cDNAs, can be directly expressed without re-synthesis and codon modification. Multi-subunit complex proteins can be expressed from polycistronic mRNAs. The marker gene elimination systems facilitate the bio-safety of the plastid transformation. Transformation of the plastid genome with economically valuable genes can now be accomplished using vectors that enable the post-transformation excision of marker genes. Removal of the plastid marker gene will facilitate public acceptance of the new transplastomic crops. The plastid transformation offered a good platform of foreign gene expression in high plants. However, this is only the first step. This technology hasnt resulted in any product commercialization because problems in the protein purification and the expression level control still need to be solved and the transplastomic plants need to be more evaluated. Plastid transformation is now routinely carried out only in tobacco while the efficiency of transformation in other plants is still too low. More experiments will be undertaken to move this technology toward practical utilization.

Cotransformation-segregation
Cotransformation includes two plasmids that target insertions at two different ptDNA locations, of which one plasmid carries a selective marker and the other a non-selected gene. Selection for the marker yields transplastomic clones that also carry an insertion of the non-selected gene. This approach was first shown in C. reinhardtii (Kindle et al., 1991; Roffey et al., 1991). When this method was used in tobacco, a cotransformation efficiency of 20% was obtained (Carrer and Maliga, 1995). The approach was developed to obtain marker-free plants that lack the antibiotic resistance gene but are resistant to the glyphosate or phosphinothricin herbicides (Ye et al., 2003). An application of cotransformation was His-tagging of an unlinked ndh gene following spectinomycin selection (Rumeau et al., 2005). These four methods are all suitable for obtaining marker-free transplastomic plants. However, during the homology-based excision and the cotransformationsegregation approaches, the final desired transplastomic line is genetically unstable. This makes it difficult to control the production of marker-free transplastomic clones. Transient cointegration to obtain marker-free plants is also difficult to control, unless selection is combined with visual aided selection by complementation of a knockout mutant (Klaus et al., 2004). By far the most convenient approach to obtain marker-free transplastomic plants is the recombinase-mediated marker excision. The problems inherent to this system are potential native sequences (pseudo sites) recognized by the recombinase and enhanced homologous recombination between non-target repeats (Lutz and Maliga, 2007). Additional enzymes of

Acknowledgements
This research was supported by the Ministry of Science and Technology of China (863 Projects) (No.

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2007AA100505 and 2005AA206150). We thank Dr. Richard R.-C. Wang (USDA-ARS, FRRL) for English editorial assistance.

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