Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.     

OBPC Symposium: Maize 2004 &; beyond—Recent advances in chloroplast genetic engineering

Summary The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrols) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.     

Elimination of deleterious mutations in plastid genomes by gene conversion

Asexual reproduction is believed to be detrimental, mainly because of the accumulation of deleterious mutations over time, a hypothesis known as Muller's ratchet. In seed plants, most asexually reproducing genetic systems are polyploid, with apomictic species (plants forming seeds without fertilization) as well as plastids and mitochondria providing prominent examples. Whether or not polyploidy helps asexual genetic systems to escape Muller's ratchet is unknown. Gene conversion, particularly when slightly biased, represents a potential mechanism that could allow asexual genetic systems to reduce their mutation load in a genome copy number-dependent manner. However, direct experimental evidence for the operation of gene conversion between genome molecules to correct mutations is largely lacking. Here we describe an experimental system based on transgenic tobacco chloroplasts that allows us to analyze gene conversion events in higher plant plastid genomes. We provide evidence for gene conversion acting as a highly efficient mechanism by which the polyploid plastid genetic system can correct deleterious mutations and make one good genome out of two bad ones. Our finding that gene conversion can be biased may provide a molecular link between asexual reproduction, high genome copy numbers and low mutation rates.     

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.     

Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.     

Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome

Applications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene beta-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to beta-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.     

The complete chloroplast genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins of chlorarachniophyte and euglenid secondary endosymbionts

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.     

Plastid genetic engineering in Solanaceae

Plastid genetic engineering has come of age, becoming today an attractive alternative approach for the expression of foreign genes, as it offers several advantages over nuclear transformants. Significant progress has been made in plastid genetic engineering in tobacco and other Solanaceae plants, through the use of improved regeneration procedures and transformation vectors with efficient promoters and untranslated regions. Many genes encoding for industrially important proteins and vaccines, as well as genes conferring important agronomic traits, have been stably integrated and expressed in the plastid genome. Despite these advances, it remains a challenge to achieve marked levels of plastid transgene expression in non-green tissues. In this review, we summarize the basic requirements of plastid genetic engineering and discuss the current status, limitations, and the potential of plastid transformation for expanding future studies relating to Solanaceae plants.     

Microfilaments and microtubules control the morphology and movement of non-green plastids and stromules in Nicotiana tabacum

Plastid stromules are stroma-filled tubular extensions of the plastid envelope membrane. These structures, which have been observed in a number of species, allow transfer of proteins between interconnected plastids. The dramatic shape of stromules and their dynamic movement within the cell provide an opportunity to study the control of morphology and motion of plastids. Using inhibitors of actin and tubulin, we found that both microfilaments and microtubules affect the shape and motility of non-green plastids. Actin and tubulin control plastid and stromule structure by independent mechanisms, while plastid movement is promoted by microfilaments but inhibited by microtubules. The presence or absence of stromules does not affect the motility of plastids. Photobleaching experiments indicate that actin and tubulin are not necessary for the bulk of green fluorescent protein (GFP) movement between plastids via stromules.     

The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

Transposition of a bacterial insertion sequence in chloroplasts

Bacterial transposable elements (IS elements, transposons) represent an important determinant of genome structure and dynamics, and are a major force driving genome evolution. Here, we have tested whether bacterial insertion sequences (IS elements) can transpose in a prokaryotic compartment of the plant cell, the plastid (chloroplast). Using plastid transformation, we have integrated different versions of the Escherichia coli IS element IS 150 into the plastid genome of tobacco ( Nicotiana tabacum ) plants. We show that IS 150 is faithfully mobilized inside the chloroplast, and that enormous quantities of transposition intermediates accumulate. As synthesis of the IS 150 transposase is dependent upon programmed ribosomal frame shifting, our data indicate that this process also occurs in chloroplasts. Interestingly, all insertion events detected affect a single site in the plastid genome, suggesting that the integration of IS 150 is highly sequence dependent. In contrast, the initiation of the transposition process was found to be independent of the sequence context. Finally, our data also demonstrate that plastids lack the capacity to repair double-strand breaks in their genomes by non-homologous end joining, a finding that has important implications for genome stability, and which may explain the peculiar immunity of the plastid to invading promiscuous DNA sequences of nuclear and mitochondrial origin.     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process

The plastid genome (ptDNA) of higher plants is highly polyploid, and the 1000-10 000 copies are compartmentalized with up to approximately 100 plastids per cell. The problem we address here is whether or not a newly arising genome can be established in a developing tobacco shoot, and be transmitted to the seed progeny. We tested this by generating two unequal ptDNA populations in a cultured tobacco cell. The parental tobacco plants in this study have an aurea (yellowish-golden) leaf color caused by the presence of a bar(au) gene in the ptDNA. In addition, the ptDNA carries an aadA gene flanked with the phiC31 phage site-specific recombinase (Int) attP/attB target sites. The genetically distinct ptDNA copies were obtained by Int, which either excised only the aadA marker gene (i.e. did not affect the aurea phenotype) or triggered the deletion of both the aadA and bar(au) transgenes, and thereby restored the green color. The ptDNA determining green plastids represented only a small fraction of the population and was not seen in a transient excision assay, and yet three out of the 53 regenerated shoots carried green plastids in all developmental layers. The remaining 49 Int-expressing plants had either exclusively aurea (24) or variegated (25) leaves with aurea and green sectors. The formation of homoplastomic green shoots with the minor green ptDNA in all developmental layers suggests that the ptDNA population in a regenerating shoot apical meristem derives from a small number of copies selected through a stochastic process.     

Plastid genome sequence of the cryptophyte alga Rhodomonas salina CCMP1319: lateral transfer of putative DNA replication machinery and a test of chromist plastid phylogeny

Cryptophytes are a group of unicellular algae with chlorophyll c-containing plastids derived from the uptake of a secondary (i.e., eukaryotic) endosymbiont. Biochemical and molecular data indicate that cryptophyte plastids are derived from red algae, yet the question of whether or not cryptophytes acquired their red algal plastids independent of those in heterokont, haptophyte, and dinoflagellate algae is of long-standing debate. To better understand the origin and evolution of the cryptophyte plastid, we have sequenced the plastid genome of Rhodomonas salina CCMP1319: at 135,854 bp, it is the largest secondary plastid genome characterized thus far. It also possesses interesting features not seen in the distantly related cryptophyte Guillardia theta or in other red secondary plastids, including pseudogenes, introns, and a bacterial-derived gene for the tau/gamma subunit of DNA polymerase III (dnaX), the first time putative DNA replication machinery has been found encoded in any plastid genome. Phylogenetic analyses indicate that dnaX was acquired by lateral gene transfer (LGT) in an ancestor of Rhodomonas, most likely from a firmicute bacterium. A phylogenomic survey revealed no additional cases of LGT, beyond a noncyanobacterial type rpl36 gene similar to that recently characterized in other cryptophytes and haptophytes. Rigorous concatenated analysis of 45 proteins encoded in 15 complete plastid genomes produced trees in which the heterokont, haptophyte, and cryptophyte (i.e., chromist) plastids were monophyletic, and heterokonts and haptophytes were each other's closest relatives. However, statistical support for chromist monophyly disappears when amino acids are recoded according to their chemical properties in order to minimize the impact of composition bias, and a significant fraction of the concatenate appears consistent with a sister-group relationship between cryptophyte and haptophyte plastids.