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1. Details of Module and its Structure

Module Detail Subject Name Botany

Paper Name Plant Genetic Engineering

Module Name/Title Transplastomic Plants and Chloroplast Engineering

Module Id Module Id

Pre-requisites Basic knowledge about plastid and its function and plant genetic engineering

Objectives To create awareness to students about chloroplast transformation and its application in improving crops traits.

Keywords Chloroplast, Chloroplast transformation, Transplastomic plants, Crop improvement, Genetic engineering

Structure of Module/Syllabus of a module (Define Topic / Sub-topic of module) Transplastomic Plants

and Chloroplast Engineering

<Sub-topic Name1>

2. Development Team

3. Role

Name Affiliation

Subject Coordinator Dr.SujataBhargava Savitribai Phule Pune University

Paper Coordinator Dr.RohiniSreevathsa National Research Centre on Plant Biotechnology, Pusa, New Delhi

Content Writer/Author (CW)

Dr.SubodhKumar Sinha National Research Centre on Plant Biotechnology, New Delhi

Content Reviewer (CR) <Dr.RohiniSreevathsa>

Language Editor (LE) <Dr. AN Latey> University of Pune, Pune

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Table of contents 1. Introduction

2. Plastid genome and genomics 3. Chloroplast Genome Sequences

4. Chloroplast transformation technology 4.1 Transformation method:

4.2 Selection marker genes 4.3 Insertion sites

4.4 Regulatory sequences

5. Advantages of Plastid Transformation

6. Manipulation of Agricultural Traits through Transplastomic Approach 6.1 Abiotic Stress Tolerance

6.2 Biotic Stress Resistance

6.2.1 Insect Pest Resistance 6.2.2 Disease Resistance 6.3 Herbicide detoxification

7. Conclusions

8. Suggested readings

Introduction

Plastids are double membrane-bound organelles found in green algae and most cell-types of plants. Depending on cell- and tissue-type, plastids are specialized for the synthesis and accumulation of various metabolites. For example, amyloplasts in roots are specialized for the storage of starch; chromoplasts (as in some fruit) are specialized for carotenoid accumulation; and chloroplasts are specialized for photosynthesis. Chloroplast is the site of photosynthesis, the process responsible to provide the primary source of food productivity. Chloroplasts also serves in evolution of evolution of oxygen, sequestration of carbon, production of starch, synthesis of amino acids, fatty acids, and pigments, and participate in sulfur and nitrogen metabolism (Verma and Daniell, 2007). The various plastid types in a plant develop from undifferentiated proplastids in meristem tissues. For instance, chloroplasts begin to differentiate from proplastids in the leaf primordium early in leaf development, and chloroplast divisions occur concomitantly with cell division and cell elongation, giving rise to roughly 100 plastids on an average in the cell. Chloroplast divisions are usually completed by the time the leaf has attained full expansion (reviewed in Goldscmidt-clermont, 1998).

Plastids of higher plants and green algae, red algae and glaucophytes are “primary plastids” that evolved via endosymbiosis between a host eukaryotic cell and a prokaryote

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most likely a cyanobacterium (McFadden, 2001; Palmer, 2003; Yoon et al., 2004). The idea of endosymbiotic evolution is supported on the basis of morphological, biochemical, and molecular phylogenetic evidences. For instance, there is considerable similarity between the transcription and translation machinery of cycanobacteria and plastids.

Phylogenetic analyses have also revealed a close relatedness between gene sequences on the cyanobacterial and plastid genomes (Raven and Allen, 2003). Both chloroplasts and cyanobacteria extract light energy by Photosystems I and II (Raven and Allen, 2003).

Last but not least, plastids are surrounded by two membranes: the outer one is probably a product of the host membrane while the inner one is derived from the ancestral prokaryotic plasma membrane. However, the issue of chloroplasts evolution from either a single (monophyletic) or several (polyphyletic) independent endosymbiotic events is still somewhat controversial. However, the vast majority of studies support a single event (Archibald and Keeling, 2002; Palmer, 2003). This event gave rise to a “primary plastid”.

However, secondary or even tertiary endosymbiosis cycles have occurred, generating

“secondary (or tertiary) plastids”. These events happened when a non photosynthetic eukaryote engulfed a cell with a primary plastid (McFadden, 2001). Secondary and tertiary plastids are found in some groups of algae, including euglenophytes, chlorarachniophytes, heterokonts, haptophytes, cryptophytes, dinoflagellates, apicomplexans and prymnesiophytes.

Plastid genome and genomics

Chloroplast genomes of higher plants have double stranded, circular, small size (up to 200 kb) DNA. The presence of chloroplast genome in numerous copies produces an amplification of approximately 10,000 copies per cell. One of the characteristic feature of chloroplast genome is its lesser tendency to recombination and retained most of the ancestral genes which makes it an excellent tool for phylogenetic and evolutionary studies (Gao et al. 2010; Hibberd et al. 1998; Ravi et al. 2008). The first restriction map of a chloroplast DNA (cpDNA) was from maize (Bedbrook and Bogorad, 1976) which showed its consistency with a circle. The size of the DNA was determined to be 155 Kbp, and it was found to contain two large Inverted Repeat (IR) regions of about 22 Kbp each, which are separated from one another by “Large Single Copy” (LSC) and “Small Single Copy”

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(SSC) regions. Chloroplasts contain multiple identical cpDNA (chloroplast DNA) copies, and range in size from 120 to 160 Kbp, depending on the species. Complete cpDNA sequences were first reported for tobacco (Nicotianatabacum) (Shinozaki et al., 1986) and liverwort (Ohyamaet al., 1986). Although the overall structure of the chloroplast genome is highly conserved among higher plants, comparative sequence analyses have revealed that some structural changes have occurred (reviewed in Palmer, 1985). These include inversions, translocations, and insertion/deletions. As far as different forms of cpDNA are concerned, there is a notion that cpDNA is a genome-sized circle that was based on early EM observations of Kolodner and Tewari (1972) showing that there is a single size class of circular cpDNA in pea, and that the length of this DNA matches the size of the genome as estimated by DNA re-association kinetics. However, linear forms were also observed in the EM. As these linear forms were smaller than genome length they were assumed to arise from breakage of the circles. This gave rise to the “Broken Circles” model of the structure of chloroplast DNA, which has served as a paradigm for over 30 years. Reports of more complex forms of cpDNA were interpreted as rare catenated molecules of genome size, still consistent with this model (Deng et al., 1989; Bendich, 1991). “Broken Circles” model was later questioned by Bendich and co-workers who provided evidence that the bulk of cpDNA is linear (Bendich, 2004; Oldenburg and Bendich, 2004). The linear molecules appear to have defined ends that reside near putative origins of replication. It is hypothesized that the linear cpDNA forms give rise to branched, multigenomic structures (“nucleoids”) during replication. These complex structures have been observed in maize and Arabidopsis by high resolution digital imaging. Subsequently, it has been thought that cpDNA replication proceeds via the formation of D-loops, Cairns intermediates and rolling circles (Kolodner and Tewari, 1975). Yet, the predominance of linear and complex branched structures suggests that this might not be the major mechanism of replication, rather the bulk of cpDNA might be replicated via recombination-dependent replication.

Most of the genes in the chloroplast genome are mainly involved in transcription and translation of the plastid genome and in photosynthesis (Douglas and Raven, 2002). The chloroplast genome contains a complete set of ribosomal RNA genes (23S, 16S, 5S and 4.5S rRNAs) and a full complement of 30 tRNA genes. These genes are similar to ones found in Escherichia coli (Sugiuraet al., 1998), consistent with the idea that the plastid

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protein synthesis machinery is prokaryotic-like in nature and based on 70S ribosomes.

Around 70 protein coding genes on the chloroplast genome are devoted to photosynthetic functions, such as genes for subunits of the various photosynthetic complexes. Yet, a large number of chloroplast genes also code for proteins necessary for protein synthesis - ribosomal subunits, RNA polymerase subunits - as well as other plastid functions, such as the ClpP1 subunit of the Clp protease. Many chloroplast genes are interrupted by at most a single intron and nearly all are transcribed as polycistronic messages, which are often modified by RNA editing and alternative splicing.

Each chloroplast consists of 50–100 copies of plastid genome and each cell consists of more than hundreds of chloroplasts making the copy number of around 10,000 per cell (Bendich, 1987). This feature enables genetic engineers to over express the transgenes of interest, thereby allowing the recombinant proteins to accumulate at high concentrations of over 10% of the total soluble proteins (Daniell, 2006).

Chloroplast Genome Sequences

The advent of new sequencing technology such as the next generation sequencing (NGS) has revolutionized the rapid sequencing of plastid genome of thousands of plants from various groups (Gao et al., 2010) resulting the sequencing of approximately 230 plastids till recently(NCBI organelle genomes). This includes members of almost all group of plant kingdom including flowering plants, and others like bryophytes, lycophytes, gymnosperms, green and red algae, photosynthetic dinoflagellate, chromalveolates, and other photosynthetic organisms. The huge amount of information generated has led us to a better understanding of phylogenetic relationships at very low taxonomic levels and also in designing strategies for efficient transplastomic technology for crop improvement.

Since chloroplasts are maternally inherited, the transgene integrated into plastid genome would potentially negate the chances of pollen escape from GM crops into environment thus offering an effective biological containment (Bansal and Sharma, 2003, Sharma et al., 2005,Clarke and Daniell, 2011, Daniell et al., 1998, Ruf et al., 2007, Svab and Malliga, 2007) which has led to perception of the safer alternative of this technology. Therefore, development of plastid transformation technology in crop plants was given considerable emphasis in the past few years.Despite of available plastid transformation technique in

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number of crop plants, such as rice (Khan and Malliga, 1999; Lee et al., 2006), tomato (Ruf et al., 2001), potato (Nguyen et al., 2005, Sidorov et al., 1999, Valkov et al., 2011), oilseed rape (Cheng-Wei et al., 2010, Hou et al., 2003), Lesquerella (Skarjinskaia et al., 2003), lettuce (Lelivelt et al., 2005), soybean (Dufourmantel et al., 2004), carrot (Kumar et al., 2004a), cotton (Kumar et al., 2004b), cabbage (Liu et al., 2007), cauliflower (Nugent et al., 2006), sugarbeet (De Marchis et al., 2009), brinjal (Singh et al., 2010), no transplastomic crops are commercially available till date. Implementation of this technology in agronomically important crops would highly beneficial to enhance food security of the burgeoning world population (Bansal and Saha, 2012).

In this module we shall study the techniques used for genetic transformation of chloroplasts and the applications of this technology in genetic improvement of crops.

Chloroplast transformation technology

Transformation method:

Biolistic approach is most popular in chloroplast transformation in which a marker gene and the gene of interest are inserted in the Escherichia coli plasmids and introduced into chloroplasts or plastids. However,Agrobacterium mediated method was also used in plastid transformation (Block et al., 1985). PEG (Polyethylene glycol)-mediated transformation method was also applied in the early days (Sporlein et al., 1991; Golds et al., 1993; O’Neillt et al., 1993) and also reported recently (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). After the first successful chloroplast transformation in Chlamydomonas reinhardttii by bombardment method (Boynton et al., 1988), this method has been exploited for chloroplast or plastid transformation which often results into higher transformation efficiency. Protocols of plastid transformation by bombardment method are available (Koop and Kofer, 1995; Lutz et al., 2006). The first stable plastid transformation was established in higher plants; Nicotiana tabacum(Daniell et al., 1990; Svab et al., 1990) soon after the chloroplast transformation in Chlamydomonas reinhardttiiwas reported. Till date, plastid transformation has been tried so many higher plants, such as Arabidopsis (Sikdar et al., 1998), rape (Hou et al., 2003),

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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).

Selection marker genes

The presence of ptDNA (plastid DNA) in many copies makes selectable marker genes 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). 16S rRNA (rrn16) gene was first to be used as selection marker in chloroplast transformation (Svab et al., 1990).

Subsequently, the aadAgene encoding aminoglycoside 3′-adenylyl transferase was also used as a selection marker gene (Goldschmidt-Clermont, 1991; Svab and Maliga, 1993).

Transformation with aadAgene resulted into dramatic improvement in the recovery of plastid transformants to a rate of, on average, about one transplastomic line in a bombarded leaf sample (Svab and Maliga, 1993). Another popular selection marker is npt II largely used in plastid transformation of tobacco with considerably low transformation efficiency (Carrer et al., 1993). A significant improvement in plastid transformation efficiency has been achieved with the highly expressed neo gene (Kuroda and Maliga, 2001b). The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT), has also been tested as a marker gene, however 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., 2001). 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).

Insertion sites

Plastid expression vectors possess left and right flanking sequences each with 1–2 kb in size from the host plastid genome, which are used for foreign gene insertion into plastid DNA via homologous recombination. ptDNA segment flanking the marker gene and the gene of interest plays very important role determining the site of insertion in the plastid

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genome. trnV-3'rps12，trnI-trnAand trnfM-trnGare three common insertion sites which were most frequently used (Maliga, 2004). The trnV-3'rps12 and trnI-trnAsites 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-trnGsite is located in the large single copy region of the ptDNA, and the gene inserted between trnfMand trnGshould have only one copy per ptDNA. Because of the high level of protein expression the pPRV series vectors commonly used in tobacco (Maliga, 2003). Several laboratories have inserted transgenes between the trnIand trnAgenes in several plant species (Daniell et al., 1998). These two tRNAs are located between the small (rrn16) and large (rrn23) rRNA subunit genes.

Regulatory sequences

Promoter sequences and 5’-UTR elements play major role in the gene expression level in plastids (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). The most commonly used promoter is the plastid rRNA operon (rrn) promoter (Prrn).The 5′-UTR and 3′-UTR sequencesflanking the transgenes confers the stability of the transgenic mRNA which in turn becomes important in protein accumulation from the transgene. The psbA/TpsbA are most commonly used 5′- and 3’- UTR (Zoubenko et al., 1994; Millán et al., 2003; Watson et al., 2004; Daniell et al., 2005; Kittiwongwattana et al., 2007).

Advantages of Plastid Transformation

Plastid transformation technique poses several beneficial features which has generated interests among researchers to choose it as an alternative tool as compared to nuclear transformation. Some of these promising advantages are:

(A) High transgene expression: owing to polyploidy nature of the plastid genome it ensures high tissue specific transgene expression and foreign protein accumulation (5–25% of total soluble protein) and also high stability of transgene.

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(B) Regulated gene expression: highly regulated transgene expression helped by well defined promoter set and expression cassettes.

(C) Transgene stacking: because of polycistronic translation mechanism there is possibility of simultaneous introduction of multiple traits (Krichevsky et al., 2010)

(D) Position effect: due to absence of high order chromatin structure in plastid DNA and transgene integration by homologous recombination process, the chances of unwanted position effects is negated. ; the homologous recombination mediated integration also facilitates generation only one type of transplastome

(E) Gene silencing: absence of DNA methylation and epigenetic gene silencing or co-suppression in plastid genes avoids the chances of gene silencing phenomena.

(F) Transgene containment: it is possible due to strict maternal transmission of plastid genes in most crop plants resulting into less ecological risk (Bock, 2001, Meyers et al., 2010, Wang et al., 2009).

However, there have been few reports indicating the evidences of plastid gene transfer to nucleus and possibility of pollen transmission to wild plants (Day, 2003, Huang et al., 2003, Timmis, 2003). Never the less, the level of transgene containment offered by plastid is always higher than nuclear transgene. Thus, plastid transformation in crop plants offers a satisfactory platform for expressing transgenes of various agronomic traits.

Manipulation of Agricultural Traits through Transplastomic Approach

Till date transplastomic plants have been established targeting agronomic traits only in model plant system tobacco and few crop plants, and commercial crops are yet to be developed through transplastomic approach. However, the beneficial features of plastid transformation in crop plants promise not only the over expression of foreign protein but also offer an opportunity to improve several agronomic traits with least environmental risks.

Abiotic Stress Tolerance

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Several genes have also been used to impart drought tolerance in plants through transplastomic approach. Yeast trehalose phosphate synthase (TPS1) gene was used to achieve drought tolerance by expressing in both nucleus and plastids of tobacco. A transplastomic plants which produced higher level (25 fold) of trehalose accumulation found to be drought tolerantwithout any pleiotropic effects as compared to nuclear transgenic plants (Lee et al., 2003). The transplastomic carrot cells expressing high level of BADH exhibited enhanced salt tolerance even in the presence of very high salt stress (Kumar et al., 2004). Fatty acid desaturase gene has also been found suitable for imparting cold tolerance tobacco plants when expressed transplastomically possibly by manipulating lipid content in vegetative and reproductive tissues (Craig et al., 2008).

Biotic Stress Resistance Insect Pest Resistance

Cry protein genes have been widely known for imparting insect resistance in crop plants, however, the phenomenon of ‘codon biasness’ of these prokaryotic genes (AT rich) in the eukaryotic nucleus (GC rich) drastically hindering the expression level. Transplastomic approaches provide an opportunity of developing resistance against this toxin at the same time with reduced ecological risk (Losey et al., 1999). For instance, expression of native BtCry1Ac gene in tobacco chloroplasts under 16S rrn promoter with chimeric ribosome binding site of rbcL and 3’ UTR of rps16 gene exhibited high (3–5% TSP) accumulation of Bt toxins in leaves (McBride et al., 1994). Leaves from transplastomic tobacco plants proved to be 100% lethal against tobacco budworm (Heliothisvirescens), cotton bollworm (Helicoverpazea) and beet armyworm (Spodopteraexigua) without developing resistance unlike Cry1A genes (Kota et al., 1999). Elevated expression of b-glucosidase (Bgl-1) in the tobacco plastids has showed protection against aphids and whiteflies due to increase in sucrose ester levels (Jin et al., 2011).

Disease Resistance

Synthetic microbial lytic peptide (MSI-99) when expressed in tobacco chloroplasts resulted into high level of peptide expression (21.5% TSP) and resistance against Pseudomonas syringae, and spores of fungal species Aspergillus and Fusarium (DeGray et al., 2001).

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MSI-99 is an antimicrobial peptide (AMP) with an amphipathic α helix that binds to phospholipids of outer membrane of bacteria and fungi and these peptides aggregate to form pores and results into bacterial lysis. Since AMPs function at high dose, transplastomic approach would be desirable. (Daniell et al., 2002). In an another example, argK gene of P. syringaepv. Phaseolicola, coding for toxin-resistant enzyme ROCT, was introduced into tobacco chloroplasts using a plastid transit peptide (pea rbcS) and Agrobacterium transformation. The transgenic plants showed high level of fungal and viral pathogen resistance.

Herbicide Detoxification

EPSPS is known to impart herbicide detoxification, therefore its expression in plastid would be promising to avoid its escape from transformed nuclear genome through pollen dispersal (Chin et al., 2003, Daniell, 1999, Daniell, 2000, Ye et al., 2001). Similarly, the bar gene expression has been demonstrated in tobacco plastids to confer efficient resistance against herbicide phosphinothricin (PPT) (Lutz et al., 2001). Other examples of herbicide resistance genes expressed in model plant tobacco include crtI gene encoding phytoene desaturase from Erwininacarotova, bxn gene encoding bromoxynil specific nitrilase from Klebsiella pneumoniae exhibiting tolerance to norflorazon and bromoxynil, protoporphrinogen IX oxidase tolerant to many bleaching type herbicides (Heifetz, 2000), recombinant 4-hydroxyphenylpyruvate dioxygenase gene for isoxaflutole (Dufourmantel et al., 2007), and mutated acetolactate synthase gene for sulfonylurea herbicides (Shimizu et al., 2008).

Conclusions

The plastid transformation technology offers a cost effective platform offoreign gene expression in high plants. Plastid geneticengineering also has become a powerful tool for basic researchin plastid biogenesis and function. The prokaryotic genescan also be expressed without codon modification.Multi-subunit complex proteins can be expressedfrom polycistronic mRNAs. Plastid transformation is routinely carriedout only in tobacco whereas the efficiency of transformation in crop plants is too low. More experimental efforts are required to move this technology toward practical utilization.

Plastid transformation technologyin wide range of plants has largely been dependent

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onavailability of chloroplast genome sequences and tissue culturemediated regeneration.

However, more than 200 chloroplast genome sequencesare now available which would definitely facilitate understandingof genome evolution in plants as well as the chloroplastgenome organization. This would further facilitate effective vector constructionfor plastid transformation in any new crop species. The broader knowledge of transgenedelivery, selection, regeneration and process of achievinghomoplasmy would be beneficial to execute this technology in crop plants from wider taxonomic groups. One of the major hurdle of plastid transformationin crop plants is the availability of well characterized technology for targetingtransgenes in proplastids, which because of its small size getsphysically damaged during biolistic transformation process (Bansal and Saha, 2012). Theavailability of Cre-lox system offers aefficient method of removal of the plastid marker genewhich would further addressthe biosafety and public acceptance of the new transplastomic crops (Corneille et al., 2001). To realize the real potential of this technology towards improving agronomic traits in commercial crops, challenges associated with it needs to be addressed.

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