Chloroplast Transformation

J.S.S. MAHAVIDYAPEETHA

SRI JAYACHAMARAJENDRA COLLEGE OF ENGINEERING,

MYSORE – 570 006

GENE TRANSFER TECHNIQUES

CHLOROPLAST TRANSFORMATION

Submitted To,

Dr. S.C. Hiremath

Submitted By,

Shuddhodana,4JC08BT0235th SEM, B T,SJCE

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INTRODUCTION:

Chloroplasts are organelles found in plant cells that capture light energy from the sun to produce free energy through a process called photosynthesis. Chloroplasts are one of the forms a plastid may take. Other plastids are for example amyloplasts for starch storage, elaioplasts for storing fat and chromoplasts for pigment synthesis and storage. These different forms are however not fixed, and plastids have the ability to (re)differentiate between these forms. All plastids, including chloroplasts, are derived from proplastids. Most plants inherit the plastids, including chloroplasts, from only one parent. Flowering plants (“angiosperms”), like sugar beet and oilseed rape, generally inherit plastids from the mother, and chloroplast genes are not carried by pollen.

Each plastid contains multiple copies of the circular plastid genome. The number of genome copies per plastid is flexible, ranging from more than 10000 in rapidly dividing cells, where plastid divisions has given rise to a large number of plastids, to 1000 or fewer in mature cells. Plastids of higher plants are semi-autonomous organelles with a small, 120 kb to 180 kb in size encoding upto 120 genes, highly polyploid genome and their own transcription and translation machinery. The challenge of plastid transformation is uniform alteration of each of the 1000 to 10000plastid DNA (ptDNA) copies in a cell, a requirement to obtain a genetically stable plant.

Plastid transformation was first achieved in 1988 in a unicellular alga, Chlamydomonas reindhartii, followed in 1990 by transformation of the plastid genome in tobacco, a higher plant. Plastid transformation has since been extended to the algae Euglena gracilis and Porphyridium sp., the moss Physcomitrella patents and at least eleven higher plant species. Plants with transformed plastid genomes are termed transplastomic plants.

TRANSFORMATION OF THE PLASTID GENOME:

The chloroplast genome typically consists of basic units of double-stranded DNA of 120 to 220 kb arranged in monomeric or multimeric circles as well as in linear molecules. The chloroplast genome generally has a highly conserved organization, with most land plant genomes having two identical copies of a 20- to 30-kb inverted repeat region (IRA and IRB) separating a large single copy (LSC) region and a small single copy (SSC) region.

Plastid transformation is typically based on DNA delivery by the biolistic process on the surface of tungsten or gold particles or occasionally by polyethylene glycol (PEG) treatment of protoplasts. This is followed by transgene integration into the chloroplast genome via homologous recombination (Fig.1) facilitated by a RecA-type system between the plastid-targeting sequences of the transformation vector and the targeted region of the plastid genome. Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination. Targeting sequences have no special properties other than that they are homologous to the chosen target site and are generally about 1 kb in size. Both flanking sequences are essential for homologous recombination. Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population of plastid genomes. If the transgene is targeted into the IR region, integration in one IR is followed by the phenomenon of copy correction that duplicates the introduced transgene into the other IR as well.

Genetic Engineering [BT-540] 11/25/2010

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Transgenes have been stably integrated at several sites within the plastid genome. Transgenes were first integrated into transcriptionally silent spacer regions. However, transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5′ or 3′ untranslated regions (UTRs) or promoters. To date, the most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the chloroplast genome. The foreign gene expression levels obtained from genes integrated at this site are among the highest ever reported. It appears that this preferred site is unique and allows highly efficient transgene integration and expression.

Chloroplast vectors may also carry an origin of replication that facilitates replication of the plasmid inside the chloroplast, thereby increasing the template copy number for homologous recombination and consequently enhancing the probability of transgene integration. oriA is present within the trnI flanking region, and this might facilitate replication of foreign vectors within chloroplasts, enhance the probability of transgene integration. This is further confirmed by the first successful Rubisco engineering obtained by integrating the rbcS gene at this site. All other earlier attempts on Rubisco engineering at other integration sites within the chloroplast genome were only partially successful. Integration of transgenes between exons of trnA and trnI also facilitates correct processing of foreign transcripts because of processing of introns present within both flanking regions.


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Fig.1. Sorting of chloroplast genomes and isolation of homoplasmic transplastomic cell lines. The initial chloroplast transformation event involves the change of only a single (or at most a few) out of several thousand chloroplast genome copies in a leaf cell. During subsequent cell and organelle divisions, the presence of high concentrations of the selecting antibiotic favours multiplication of chloroplasts containing transformed genomes, whereas chloroplasts harbouring only wild-type genomes may be eliminated effectively. However, individual chloroplasts may still contain a mixed population of wild-type and transformed chloroplast genome molecules (intraorganellar heteroplasmy). In additional rounds of plant regeneration on selective medium, gradual sorting out of residual wild-type genomes is achieved, eventually leading to cells with a homogeneously transformed population of chloroplast genomes commonly referred to as ``homoplasmic'' or ``homochloroplastic''.

Plastid transformation is based on homologous recombination between the transforming DNA and the plastid genome (Fig. 2), coupled with selective enrichment of the transformed plastid genomes that acquire resistance to antibiotics in a cell culture. Key to the recovery of transformed plastid (transplastomic) clones is the choice of selective markers. The most commonly used selective antibiotic is spectinomycin; and most plastid transformation vectors carry an aadA gene that confers resistance to spectinomycin and streptomycin. Kanamycin is also a suitable selective agent. Plastid transformation vectors are E. coli plasmid derivatives with cloned ptDNA sequences (1–2 kb) that flank both sides of a selectable marker gene and cloning sites. The ptDNA sequences serve as targeting regions to direct integration into the plastid genome (Fig. 2). The plastid vector is propagated in E. coli, and then introduced into plastids where the marker gene and the gene of interest integrate in the targeted region by two homologous recombination events. The E. coli vector part does not carry a plastid replication origin and is subsequently lost. Plastid targeting sequences do not have special properties and may derive from any part of the plastid genome.


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Fig. 2. Introduction of a useful gene into the plastid genome linked to a selective marker. The useful gene is cloned next to a selective marker in a transformation vector. Plastid genome segments included in vector are marked as left and right targeting regions (LTR, RTR), respectively. Transformed plastid genome is formed by two recombination events via homologous targeting sequences.

DNA DELIVARY:

A critical development for the progress of organelle biology was the biolistic DNA delivery enabling transformation of Chlamydomonas chloroplasts, yeast mitochondria, and higher plant chloroplasts. The original gun with particles accelerated by a gunpowder charge was quickly replaced by a cleaner version, using highpressure He gas as propellant. The particle gun remains unchanged, except that an adaptor was introduced to simultaneously accommodate seven macrocarriers (Hepta adaptor; Biorad, Hercules, CA). Tungsten or gold particles work equally well. Biolistic delivery is the system of choice for most laboratories, as manipulation of leaves, cotyledons, or cultured cells in tissue culture requires less experience than the alternative PEG treatment of protoplasts.

PEG-mediated transformation of plastids requires enzymatically removing the cell wall to obtain protoplasts, then exposing the protoplasts to purified DNA in the presence of PEG. The protoplasts first shrink in the presence of PEG, then lyse due to disintegration of the cell membrane. Removing PEG before the membrane is irreversibly damaged reverses the process. PEG treatment was first used to test transient expression of GUS(-glucuronidase) in chloroplasts, then for stable transformation of the plastid genome.

Additional approaches for DNA delivery to plastids have also been tried. The first attempt at plastid transformation used an Agrobacterium binary transformation vector. We now understand that sophisticated nuclear targeting of the transferred DNA makes use of Agrobacterium for plastid transformation a challenge. Microinjection, which looks promising for transient gene expression, has not yet yielded stable transplastomic clones.

SELECTABLE MARKERS FOR PLASTID TRANSFORMATION:

Primary Positive SelectionPrimary markers are suitable for selectively amplifying a small number of transformed

ptDNA copies. Currently known primary markers are resistance to spectinomycin, streptomycin, and kanamycin, which inhibit protein synthesis on prokaryotic-type plastid ribosomes. These antibiotics inhibit greening, cell division, and shoot formation in plant culture. Therefore, greening, faster proliferation, and shoot formation were used to identify transplastomic clones on a selective medium. The first transplastomic clones were obtained by spectinomycin selection. Because spectinomycin allows slow proliferation of nontransformed plant cells it was assumed that the choice of a drug that enables such


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“nonlethal” selection is important to recover transplastomic clones. However, transplastomic clones were soon identified by kanamycin selection using an antibiotic concentration that is considered “lethal”. Thus, slow proliferation of nontransformed cells on a selective medium is not an essential feature of the selection scheme.

Initial transformation vectors carried a plastid 16S rRNA (rrn16) gene with point mutations that prevent binding of spectinomycin or streptomycin to the 16S rRNA. The rrn16 target site mutations are recessive, and were 100-fold less efficient than the currently used dominant aadA gene. Streptomycin resistance encoded in the rps12 ribosomal protein gene was also included in an early vector. Unexplored plastid mutations that could be used in vectors are lincomycin resistance, encoded in the 23S rRNA, and triazine resistance, encoded in psbA. Plastid mutations conferring resistance to spectinomycin, streptomycin, and lincomycin were also described in Solanum nigrum. Solanum nigrum vectors with spectinomycin resistance (rrn16) and streptomycin resistance (rps12) markers were used to transform tobacco plastids.

More efficient primary plastid markers are chimeric genes in which the coding segment of a bacterial antibiotic detoxifying enzyme is expressed from plastid signals. The aadA gene encodes aminoglycoside 3′′adenylyltransferase (AAD) also called aminoglycoside nucleotidyltransferase [ANT(3′′)-I] .AAD inactivates spectinomycin and streptomycin and was used to select transplastomic clones in Chlamydomonas and tobacco. The neo (aph(3′)IIa) gene encodes neomycin phosphotransferase II [NPTII; APH(3′)-II], and was used to select transplastomic clones in tobacco. The aphA-6 gene encodes aminoglycoside phosphotransferase or APH(3′)-VI and was used to select transplastomic clones by kanamycin and amikamycin resistance in Chlamydomonas and by kanamycin resistance in tobacco.

Another potentially useful marker is a plant nuclear gene encoding betaine aldehyde dehydrogenase (BADH), which confers resistance to the toxic compound betaine aldehyde (BA). BA selection is supposedly 25-fold more efficient than spectinomycin selection. This claim has triggered research in other laboratories to confirm the advantage of selecting transplastomic clones by BA resistance.

Secondary Positive SelectionUse of secondary selective markers is dose dependent; they are not suitable to select

transplastomic clones when only a few ptDNA copies are transformed, but will confer a selective advantage when most genome copies are transformed. Examples for secondary markers are genes that confer resistance to the herbicides phosphinothricin (PPT) or glyphosate or to the antibiotic hygromycin [based on expression of the bacterial hygromycin phosphotransferase gene].

Negative SelectionThe ability to identify loss-of-function of a conditionally toxic gene forms the basis of

negative selection. A negative selection scheme in plastids utilizes the bacterial cytosine deaminase (CD) enzyme encoded in the codA gene. CD catalyzes deamination of cytosine to uracil, enabling use of cytosine as the sole nitrogen and pyrimidine source. CD is present in prokaryotes and in many eukaryotic microorganisms, but is absent in higher plants. 5-fluorocytosine is converted to 5-fluorouracil, which is toxic to cells. This negative selection scheme was utilized to identify seedlings on 5-fluorocytosine-medium from which codA was removedby the CRE-loxP site-specific recombinase.

Combination of Visual and Selective Markers


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A method is developed for rapidly identifying transplastomic sectors using pigment-deficient tobacco knockout plants as recipients. In the knockout plants, an antibiotic resistance gene (aadA) replaces a plastid gene that causes chlorophyll deficiency. The transformation vector carries the photosynthetic gene to restore green pigmentation and a second antibiotic resistance gene to favor maintenance of transformed plastids. Homoplastomic sectors and plants can be readily identified by the green color, a strategy that significantly reduces the time required to obtain homoplastomic plants.

EXCISION OF SELECTABLE MARKER GENES:

Most of the studies involving plastid transformation have utilized antibiotic resistance gene for the recovery of transformed plastomes, but introducing such crops into the food chain may be a cause of concern. Strategies have been developed to eliminate antibiotic resistance genes after transformation, including homology-based excision via directly repeated sequences, excision by phage site-specific recombinases, transient co-integration of the marker gene, and cotransformation-segregation.

Avariant of homology-based marker excision technology enabled direct identification of marker-free plants by herbicide resistance. The vector used for plastid transformation carried the aadA gene disrupting the herbicide resistance gene. The primary transplastomic clones were selected by spectinomycin resistance. Marker-free herbicide-resistant derivatives were identified after excision of the aadA marker gene by homologous recombination within the overlapping region of the N-terminal and C-terminal halves of the herbicide resistance gene. Excision of the aadA gene led to reconstitution of an entire herbicide resistance gene and expression of the Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase enzyme that conferred resistance to sulcotrione and isoxaflutole herbicides. A second variant of this approach facilitated visual tracking of homology-based marker excision by creation of a pigment-deficient zone due to the loss of a plastid photosynthetic gene rbcL.

So far two recombinases have been tested for plastid marker gene excision, the Cre and the Int. The Cre enzyme derives from the P1 bacteriophage and excises target sequences flanked by directly oriented 34-bp loxP sites . Accordingly, the marker gene is flanked in the transformation vector by two directly oriented loxP sites and DNA introduction is followed by selection for the marker gene until the homoplastomic state is achieved (Fig. 3a).When removal of the marker gene is desired, a Cre gene is introduced into the plant nucleus. The encoded CRE enzyme is plastid-targeted (Fig.3b). The Cre gene may be introduced into the plant nucleus by three methods: (1) stable transformation of the nucleus using an Agrobacterium binary vector; (2) by pollination; (3) or by transiently expressing Cre from T-DNA by agroinfiltration.


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Fig. 3. Plastid transformation and excision of the marker gene by the CRE-loxP site-specific recombination system.(a) Homologous recombination between the plastid genome (ptDNA) and plastid transformation vector (arrows) yields transformed plastid genome (TP1-ptDNA). Plastid vectors target insertion of the marker gene (m) and gene of interest (goi) via the left (L) and right (R) targeting sequences. Two loxP sites (open triangles) flank the marker gene (m is floxed) in T1-ptDNA to facilitate its removal by CRE, the P1 phage site-specific recombinase.(b) Plastid-targeted CRE is expressed from a nuclear gene and excises floxed marker gene to yield marker-free TP2-ptDNA transplastome.

The second site-specific recombinase, Int, appeared to be a better choice for the aadA marker gene removal when flanked with directly oriented nonidentical phage attP (215 bp) and bacterial attB (54 bp) attachment sites, which are recognized by Int recombinase. Efficient excision of the marker gene was shown after transformation of the nucleus with an int gene encoding plastid-targeted Int.

Alternatively, a transient co-integrative vector may even be used to avoid the integration of selectable marker genes. The marker gene in the vector in this case is placed outside the targeting region, as shown in Fig. 4. First, the transplastomic plants are selected by kanamycin resistance, that allows cointegrate formation through the left (LT) or right (RT) targeting regions.The example in Fig. 4 shows cointegration via LT. Note that LT and RT are directly repeated sequences (R1, R2) in the cointegrate symbolized by arrows. The cointegrate structures are inherently unstable: they either became stable by a second recombination event, that results in the loss of the marker gene (via R2 in Fig. 4; event #2a), or are lost by recombination via the directly oriented LT (R1) repeats restoring the wild-type ptDNAstructure (event #2b in Fig. 4). To allow formation of marker-free plants, selection for kanamycin resistance is stopped and the marker free plants are identified by screening. Combining the procedure with visually aided selection greatly accelerates obtaining marker-free transplastomic plants.

The cotransformation-segregation approach involves transformation with two plasmids that target insertions at two different ptDNA locations: one plasmid carries a selective marker and the other a nonselected gene. Selection for the marker yields transplastomic clones that also bear an insertion of the nonselected gene. The prospect of the approach was first shown in C. reinhardtii. Interestingly, when the approach was tested in tobacco, a cotransformation efficiency of 20% was obtained even though tobacco has a greater number of chloroplasts. An application of cotransformation was His-tagging of an unlinked ndh gene following spectinomycin selection.


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Fig. 4. Marker-free plants by selection for a transiently cointegrated marker gene. Shown is the vector with the left (LT) and right (RT) targeting regions flanking the gene of interest (goi) and the kanamycin resistance marker gene (aphA) outside the targeting region. Recombination via LT may lead to either a marker-free TP1 transplastome by a second recombination event via RT (#2a) or reversal of cointegration yielding a vector and wt-ptDNA (#2b).

REPORTER GENES USED IN PLASTIDS:

The E. coli GUS (-glucuronidase), chloramphenicol acetyl transferase, and the Aequorea victoria GFP (green fluorescent protein) are reporter enzymes that allow tracking gene expression, but do not confer a selective advantage or disadvantage to plastids. GUS enzymatic activity expressed in chloroplasts has been measured using fluorogenic assays and visualized by histochemical staining. GFP is a visual marker, allowing direct imaging of the fluorescent gene product in living cells. Its chromophore forms autocatalytically in the presence of oxygen and fluoresces green when absorbing blue or UV light. GFP has been used to detect transient gene expression and stable transformation events in chloroplasts. GFP was fused with the aadA gene product (AAD) to be used as a bifunctional visual and selective (spectinomycin resistance) marker gene.

IMPLIMENTATION OF PLASTID TRANSFORMATION IN DIFFERENT CROP SPECIES:

Although plastid transformation in higher plants was achieved in 1990, it is routinely done only in tobacco. Plastid transformation has also been successful in two other solanaceous species, potato and tomato. Plastid transformation in Arabidopsis thaliana and the related Brassica napus and Lesquerella fendleri was feasible but inefficient. Economically important crops such as carrot, cotton, and soybean regenerate in vitro through somatic embryogenesis. In such crops, transformation of the plastid genome was achieved through somatic embryogenesis by bombarding embryogenic non-green cells or tissues. The first stable plastid transformation of embryogenic cell cultures and somatic embryogenesis was established in carrot. Plastid transformation of embryogenic cultures in rice could also be readily obtained. Rice plants regenerated from the transformed culture were heteroplastomic , suggesting that only refining the tissue culture system is required to obtain homoplastomic plants. Spectinomycin resistance (aadA) was the marker of choice in each species, except rice, in which streptomycin resistance was used to select transplastomic clones. Spectinomycin selection in cereals is not an option because rice and maize plastid rRNAs naturally have the mutations that prevent spectinomycin binding.

APPLICATIONS IN BASIC SCIENCE:

Gene KnockoutsTargeted knockout of plastid genes involves construction of a transformation vector in

which a selectable marker replaces the target gene in a larger ptDNA fragment (Fig. 5). Selection for antibiotic resistance results in replacement of the target gene with the selective marker in the ptDNA. Positively identifying essential plastid genes has been problematic since attempts to obtain homoplastomic ycf1, ycf2, and clpP1 knockout lines by targeted insertion of a selective marker have failed. Evidence for an essential role for the ClpP1


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protease subunit was obtained by the CRE-loxP site-specific recombination system. The clpP1 gene in the plastid genome was first flanked with directly oriented loxP sites (floxed). A CRE gene was then introduced into the nucleus by pollination. The nuclear-encoded, plastid-targeted CRE entered the plastids and excised the floxed clpP1 copies. Loss of the clpP1 gene product, the ClpP1 protease subunit, led to ablation of the shoot system of plants, suggesting that ClpP1-mediated protein degradation is essential for shoot development.

Fig. 5. Targeted deletion of a plastid gene (G2) by replacement with marker gene (M1) in vector.

OverexpressionThe advantage of over expression as a research tool was shown by the unexpected

discovery of site-specific RNA editing trans-factors based on over-expression of an edited RNA segment and a clP1-specific mRNA maturation factor based on over expression of the clpP1 5′-UTR in a chimeric transcript. Over expression was used to probe the plastid accD function by replacing the weak accD promoter with the strong rrn promoter. Another example for over expression involved relocating the nuclear anthranilate synthase gene to plastids to boost tryptophan production .

TranscriptionThe field of plastid gene transcription also benefits from plastid transformation. The

plastid rpoA and rpoB tobacco knockout plants played a critical role in recognizing that the plastid-encoded plastid RNA polymerase (PEP) and the nuclear-encoded phage-type RNA polymerase (NEP) transcribe distinct groups of genes. Reporter genes expressing GUS and GFP have been utilized to identify PEP and NEP promoter elements. Of the PEP promoters, dissection in vivo was reported for the blue-light-regulated psbD promoter, the rRNA operon PEP promoter, the rbcL promoter, and the psbA promoter. Of the promoters recognized by the NEP, the clpP1 and atpB promoters have been subjected to in vivo dissection.

RNA EditingRNA editing in plastids involves posttranscriptional C to U nucleotide conversions.

Plastid transformation has been an important tool in studying RNA editing in chimeric transcripts consisting of a series of small mRNA segments transcriptionally fused with a reporter gene. Sequences required for RNA editing are contained in a small segment; RNA editing depends on site specific depletable trans-factors; if a site is not edited in species it is an indication that the capacity to edit the site is absent unless the species has sites that share specificity factor(s) with the heterologous site.

PhotosynthesisBecause many photosynthetic genes are encoded in the plastid genome, plastid

transformation is a necessary tool to probe and improve photosynthesis. Studies in higher plants focused on photosynthetic gene knockouts and Rubisco engineering. Rubisco in higher


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plants is composed of plastid encoded large and nucleus-encoded small subunits (LS and SS, encoded in the plastid rbcL and nuclear rbcS genes, respectively).

EvolutionDuring evolution most of the 3000 genes encoded in the genome of the

photosynthetic endosymbiont migrated to the nucleus. Plastid transformation enabled addressing experimentally the mechanistic details of gene transfer. The frequency of DNA transfer from plastids to the nucleus was tested by incorporating a nuclear kanamycin resistance gene in the plastid genome, then selecting for a transfer event to the nucleus by selecting for expression of a kanamycin resistance gene. The frequencies of transfer were 1 in 16,000 in the seed progeny and significantly lower (1 in 5,000,000) in somatic cells. Of course, acquiring a nuclear promoter by a transferred plastid gene is several orders of magnitude less likely than expressing a plastid gene that brings along its own nuclear promoter. The feasibility of relocating a plastid gene to the nucleus was also tested.

APPLICATIONS IN BIOTECHNOLOGY:

Agronomic TraitsIn field crops, the most common transgenic traits are resistance to insects and

herbicides expressed from nuclear genes. Both types of genes have been successfully engineered for plastid expression. Expression of the B.t. insecticidal protein from nuclear genes required construction of synthetic, codon-modified genes to improve translation, protect the mRNA from degradation, and prevent early translation termination. In contrast, the B.t. insecticidal protein genes were expressed in plastids from bacterial coding segments. Commercially useful versions of commonly used herbicide resistance genes are also available. Tobacco plants with some degree of glyphosate tolerance were obtained by overexpression of the sensitive form of 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) target enzyme in plastids. Field-level tolerance to glyphosate was obtained by expression in plastids of prokaryotic EPSPS genes; the required protein levels were higher (5% of TSP) than when EPSPS was expressed from nuclear genes. An interestingly split EPSPS gene was developed with gene containment in mind, with one half of the protein encoded in a nuclear gene and the second half in a plastid gene. Intein trans-splicing resulted in reconstituting the herbicide-resistant EPSPS enzyme in plastids. Pollen transmission from this crop can lead to the transfer of only part of the herbicide resistance gene, which is insufficient to confer glyphosate tolerance. Field-level tolerance to herbicides containing PPT as an active ingredient was obtained by expressing bar in the plastid genome.

Biopharmaceutical BioreactorsSeveral chloroplast-derived biopharmaceutical proteins have been reported. Stable

expression of a pharmaceutical protein in chloroplasts was first reported for GVGVP, a protein-based polymer with medical uses such as wound coverings, artificial pericardia, and programmed drug delivery. Human ST (hST), a secretory protein, was expressed inside chloroplasts in a soluble, biologically active and disulfide-bonded form. The key use of hST is in the cure of hypopituitary dwarfism in children; additional indications are treatment of Turner syndrome, chronic renal failure, and human immunodeficiency virus wasting syndrome. Another important therapeutic protein that comprises approximately 60% of the protein in blood serum is HSA, prescribed in multigram quantities to restore blood volume in trauma and other clinical conditions. Early attempts at expressing HSA have achieved inadequately low levels of HSA (0.2% of tsp) in nuclear transgenic plants. On the other hand,


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in chloroplast transgenic plants, expression levels of up to 11.2% have been observed. The type I IFNs are part of the body’s first line of defence against viral attack and also invasion by bacterial pathogens, parasites, tumor cells, and allogeneic cells from grafts. IFNa2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. Transgenic IFNa2b had comparable in vitro biological activity to commercially produced PEG-Intron when tested for its ability to protect BHK cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect assay and to inhibit early stage human immunodeficiency virus infection in HeLa cells. Another therapeutic protein expressed in chloroplasts is human IFN-g. In a bioassay, the chloroplast-produced human IFN-g offered complete protection to human lung carcinomas against infection by the EMC virus.

Expression of Recombinant ProteinsTo meet the demands for production capacity of recombinant proteins, there is

significant interest in plant-based production of vaccines, antibodies, and industrial enzymes. Transgene expression in tobacco plastids reproducibly yields protein levels in the 5% to 20% range. A salient feature of plastid expression is the importance of post-transcriptional regulation; from the same promoter proteins may accumulate in a 10,000-fold range. Since all codons are relatively frequently used, codon optimization yields only a modest 2- to 3-fold increase in protein accumulation levels. Transcripts derived from genes of diverse sources were stable in plastids, including bacterial genes with relatively high levels of adenine and thymine (high-AT)and guanine and cytosine (high-GC), synthetic mRNAs, and plant and human cDNAs. This suggests the compatibility of the plastid’s RNA degradation machinery with mRNAs from diverse sources, avoiding the need to construct synthetic genes for plastid expression. Furthermore, there is no protein glycosylation in plastids.

Marker Gene EliminationThe interest in developing marker elimination systems for plastids was driven by

regulatory concerns to avoid releasing antibiotic resistance genes in transplastomic crops, the desire to reuse the relatively few available plastid marker genes, and the metabolic burden imposed by expressing marker genes. Three systems are available for marker gene elimination. The first system relies on the loop-out of the marker gene through directly repeated sequences. This system is practical only in exceptional cases, when introducing secondary markers, such as herbicide resistance genes, is the desired objective. A second approach involves cotransforming two independently targeted plastid transgenes and segregating out the ptDNA with the marker gene at the heteroplastomic stage. The third and most efficient approach uses vectors with floxed marker genes, which can be removed with the CRE site-specific recombinase. Although convenient vectors with floxed marker genes have not yet been reported for the introduction of passenger genes, the feasibility of the approach was shown by excising aadA, codA and clpP1 genes. Important for the application of CRE in plastids is that no detrimental ptDNA rearrangements persist once CRE is removed.

Containment by Plastid LocalizationTransgene flow is a problem within a species, when pollen from one stand of the crop

finds its way into another stand, or when transgenes are incorporated in weedy relatives. Effectively controlling intraspecific transgene flowin species with a strict maternal inheritance of plastids can be achieved by incorporating the transgenes in the plastid genome. Examples for species with strict maternal inheritance of plastids are Zea mays, Glycine max, Oryza sativa, and Arabidopsis thaliana, a group to which two thirds of the angiosperm species belong.


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ADVANTAGES OF PLASTID TRANSFORMATION:

Chloroplasts are members of a class of organelles known as plastids and are found in plant cells and eukaryotic algae. As the site of photosynthesis, chloroplasts are the primary source of the world’s food productivity and they sustain life on this planet. 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. Chloroplasts are generally considered as derivative of a cyanobacterial ancestor that was captured early during the evolution of a eukaryotic cell.However, the chloroplast genome is considerably reduced in size as compared to that of free-living cyanobacteria, but the genomic sequences that are still present show clear similarities. Land plant chloroplast genomes typically contain 110 to 120 unique genes, whereas cyanobacteria contain more than 1,500 genes. Many of the missing genes are present in the nuclear genome of the host.

In most angiosperm plant species, plastid genes are maternally inherited, and therefore, transgenes in these plastids are not disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing lower environmental risks. This biological containment strategy is therefore suitable for establishing the coexistence of conventional and genetically modified crops. Cytoplasmic male sterility (CMS) presents a further genetic engineering approach for transgene containment. Furthermore, plant-derived therapeutic proteins are free of human pathogens and mammalian viral vectors. Therefore, plastids provide a viable alternative to conventional production systems such as microbial fermentation or mammalian cell culture.

Another advantage of plastid transformation is the ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein) when the transgene is stably integrated. This is due to the polyploidy of the plastid genetic system with up to 10,000 copies of the chloroplast genome in each plant cell, resulting in the ability to sustain a very high number of functional gene copies. Furthermore, sitespecific integration into the chloroplast genome byhomologous recombination of flanking chloroplast DNA sequences present in the chloroplast vector eliminates the concerns of position effect, frequently observed in nuclear transgenic lines. Other advantages seen in chloroplast transgenic plants include the lack of transgene silencing despite the accumulation of transcripts at a level 169-fold higher than in nuclear transgenic plants and accumulation of foreign proteins at levels up to 46% of total leaf protein.

Chloroplast genetic engineering also offers the unique advantage of transgene stacking, i.e. simultaneous expression of multiple transgenes, creating an opportunity to produce multivalent vaccines in a single transformation step. Several heterologous operons have been expressed in transgenic chloroplasts, and polycistrons are translated without processing into monocistrons. Moreover, foreign proteins synthesized in chloroplasts are properly folded with appropriate posttranscriptional modifications, including disulfide bonds and lipid modifications. This article is focused on the various components of vectors used for stable protein production in transgenic chloroplasts.


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REFERENCES:

Ralph Bock (2001). Transgenic Plastids in Basic Research and Plant Biotechnology. J. Mol. Biol. (2001) 312, 425-438.

Pal Maliga (2005). New vectors and marker excision systems mark progress in engineering the plastid genome of higher plants. First published as an Advance Article on the web 4th November 2005

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Chloroplast Transformation

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