The concept of chloroplast transformation, conceived in the mid-80s [23
], has recently blossomed into a safe and environmentally friendly technology [8
]. When the first transgenes were introduced via the chloroplast genome, it was believed that foreign genes could be inserted only into transcriptionally silent spacer regions, amidst divergent chloroplast genes [26
]. However, Daniell et al
] advanced the concept of inserting transgenes into functional operons and transcriptionally active spacer regions. This approach facilitated the insertion of multiple genes under the control of a single promoter, enabling the coordinated expression of transgenes [13••
]. Earlier reports, based on in vitro
studies of chloroplast mutants, established a definite requirement for the processing of dicistrons to monocistrons before translation [28
]. To test this hypothesis, multiple transgenes were inserted into the rRNA operon of chloroplast genomes to study their transcription, RNA processing and translation. Contrary to previous reports, the following examples unequivocally demonstrate that polycistrons are efficiently translated in transgenic chloroplasts without any requirement for RNA processing. The fact that several foreign proteins are synthesized in large quantities without any detectable monocistrons support this conclusion.
Expression of a protein-based biomedical polymer as a dicistron in transgenic chloroplasts demonstrated, for the first time, the potential of this technology to engineer biopharmaceuticals [31••
]. Recently, human serum albumin (HSA), expressed under the regulation of the optimal chloroplast ribosome-binding site (GGAGG), could not be easily detected (<0.02% tsp) in transgenic chloroplasts. In the past, the same regulatory sequence has resulted in accumulation of large quantities of several other foreign proteins (up to 21% tsp) [27•
]. HSA was, however, successfully hyper-expressed in transgenic chloroplasts as a dicistron or polycistron, by manipulating the 5′ and 3′ regulatory sequences of the transgene (A Fernandez-San Millan, A Mingo-Castel, H Daniell, unpublished results) [33
]. HSA accumulated in such large amounts that inclusion bodies formed and increased the size of transgenic chloroplasts (). HSA inclusion bodies were readily purified by simple centrifugation and solubilized to functional monomers. Regulatory sequences used in this study should serve as a model system for enhancing the expression of foreign proteins that are highly susceptible to proteolytic degradation and in addition should provide major advantages in purification. This study reports the highest level of pharmaceutical protein ever observed in transgenic plants. This is the first report to provide direct evidence for translation of transgene polycistrons, without any requirement for processing to monocistrons. Also, this study identifies a heterologous untranslated region (UTR) that could be used in non-green plastids, free of nuclear control. Searches for such non-green UTRs have been elusive so far.
Figure 1 Examples of the highest level of transgene expression. Electron micrographs of the hyper-expression of foreign proteins in transgenic chloroplasts. (a) Inclusion bodies of HSA, the most widely used intravenous protein in human therapies. (b,c) Immunogold-labeled (more ...)
To combat a disease like cholera that often assumes epidemic proportions and poses a threat as an agent of bioterrorism, there is a need for producing vaccines on an agricultural scale. Therefore, cholera toxin β subunit (CTB) was expressed in transgenic chloroplasts as a dicistron. As the quaternary structure and disulfide bonds of many pharmaceutical proteins are essential for their function, we demonstrated, using CTB, the assembly of functional oligomers in transgenic chloroplasts. Expression of the native β subunit gene (ctx
B) was 410-fold higher than in nuclear transgenic plants and there were no pleiotropic effects, in contrast to nuclear transgenic plants that showed stunted growth [16•
]. Western blot analysis and enzyme-linked immunosorbant assay (ELISA) showed that several independent transgenic lines expressed the same amount of CTB, except for physiological variations [16•
]. Engineering CTB in transgenic chloroplasts, along with recent success in the chloroplast transformation of edible crops and the availability of plant-derived selectable markers, augur well for producing edible vaccines in transgenic chloroplasts on a cost-effective basis [16•
Chloroplast transformation has also been employed to confer resistance to biotic and abiotic stresses. Expression of an antimicrobial peptide, MSI-99, as a dicistron in transgenic chloroplasts was shown to inhibit the growth of several plant pathogens, including Pseudomonas syringae, Aspergillus flavus
, Fusarium moniliformae, Verticillium dahliae
and the multidrug-resistant human pathogen Pseuodomonas aeruginosa,
when tested using in planta
and in vitro
]. Lysis of transgenic chloroplasts at the site of infection resulted in high-dose release of the antimicrobial peptide (800 μg MSI-99, inhibitory concentration 1 μg MSI-99 for 1000 bacterial cells or fungal spores). In another recent report, the integration of a yeast trehalose-6 phosphate synthase (TPS) gene as a dicistron in transgenic chloroplasts was shown to confer drought tolerance, as evidenced by growth of transgenic plants on 6% polyethylene glycol and ability to rehydrate after dehydration [15•
]. Whereas nuclear transgenic plants accumulating trehalose in the cytosol showed stunted growth, sterility and other pleiotropic effects, chloroplast transgenic plants showed normal growth and physiology and no pleiotropic effects (). Chloroplast transgenic plants showed 16 699% more tps
1 transcripts than the best nuclear transgenic plants, alleviating the possibility of gene silencing in transgenic chloroplasts ().
Figure 2 Alleviation of pleiotropic effects. Comparison of the phenotypic effects of trehalose accumulation in the cytosol and chloroplasts of transgenic plants. (a) Untransformed wild type (1); nuclear transgenic plants from different, independent transgenic (more ...)
Figure 3 Elimination of gene silencing. Northern blot analysis of nuclear and chloroplast transgenic plants expressing the trehalose phosphate synthase (tps1) gene. (a) Steady-state transcript levels of tps1: (1) untransformed wild type; (2) and (3) highly expressing (more ...)
Perhaps the most significant accomplishment, which has made chloroplast transformation technology safe, is the use of a plant-derived selectable marker, betaine aldehyde dehydrogenase (BADH), to obtain chloroplast transgenic plants by expression of a dicistron [18••
]. The selection process involves conversion of toxic betaine aldehyde to glycine betaine by BADH; glycine betaine also serves as an osmoprotectant. The BADH gene derived from spinach not only eliminates the need for the use of antibiotic resistance genes but is also 25-fold more efficient than antibiotic resistance genes, exhibiting rapid regeneration of transgenic shoots within two weeks. These developments should help to allay public concerns and make genetically modified foods more acceptable.
Ever since chloroplast technology was conceived, it was anticipated that the prokaryotic nature of the organelle should allow the expression of bacterial operons. This promise was realized when expression of the B. thuringiensis cry
2Aa2 operon in transgenic chloroplasts led to the formation of insecticidal crystals () [13••
]. The 4.0 kb operon consists of three genes, with cry
2Aa2 being the distal gene. The open reading frame, orf
2, immediately upstream of the gene codes for a putative chaperonin that is necessary for folding the protein into cuboidal crystals (that are resistant to proteolytic degradation). Expression of the operon in transgenic chloroplasts resulted in the accumulation of Cry2Aa2 protein at 46.1% of tsp, even in senescing bleached old leaves. Such high levels of insecticidal protein were instrumental in combating insects that are normally difficult to control, including the 10-day old cotton bollworm and beet armyworm. Observed hyper-expression of Cry2Aa2 protein argues against any possibility of gene silencing in transgenic chloroplasts.
The possibility of expressing a pharmaceutical protein, which involves multiple genes, has been explored using the Guy’s 13 monoclonal antibody. This antibody against the surface protein of Streptococcus mutans
, which is the causative agent of dental caries, was successfully expressed and properly assembled in transgenic chloroplasts [14
]. This is the first demonstration of expression of a multisub-unit foreign protein that is assembled with disulfide bridges. Application of Guy’s 13 monoclonal antibody to the dental surface prevented recolonization of the bacterium for up to two years [39
]. This multisubunit antibody has been expressed via the nuclear genome by generating independent transgenic lines, followed by subsequent breeding [40
]. For commercial application, however, expression levels should be increased further in nuclear transgenic plants.
Phytoremediation is evolving as a safe technology to address the increasing problem of the pollution of soil and water bodies. One of the most toxic pollutants that threatens our health and ecosystem is mercury. In the environment, mercury is rapidly methylated by bacteria producing a 10-fold more toxic organomercurial, owing to its ability to cross lipid membranes [41
]. Over 90% of methylmercury is absorbed in blood compared with only 2% of inorganic mercury, causing neurological degeneration in birds, mammals and humans. In photosynthetic organisms, mercury inhibits the oxygen-evolving enzyme (OEE) complex, binds to thylakoid membranes [42
] and removes EP33 (one of the proteins of the OEE complex [43
]). Mercury reduces the variable fluorescence (which provides a measure of photosynthetic efficiency) owing to additional inhibitory sites on the donor side of photosystem II, causing damage to the light-harvesting complexes and structural changes in the antenna pigments that affect the primary photochemistry; mercury also inhibits plastocyanin [44
]. Nuclear codon optimized mer
A (mercury ion reductase) and mer
B (organomercurial lyase) genes were used to obtain transgenic plants that are resistant to mercury and organomercurials, respectively (up to 10 μM) [45•
]. The low level of tolerance observed might result from the low levels of nuclear expression, compounded by the fact that these enzymes were not targeted to chloroplasts, where mercury is most toxic, requiring continuous detoxification. Therefore, the mer
operon has been expressed via the chloroplast genome to overcome these problems [46•