Transgenic plastids in basic research and plant biotechnology

Facile methods of genetic transformation are of outstanding importance for both basic and applied research. For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome. More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast. The possibility to directly manipulate chloroplast genome-encoded information has paved the way to detailed in vivo studies of virtually all aspects of plastid gene expression. Moreover, plastid transformation technologies have been intensely used in functional genomics by performing gene knockouts and site-directed mutageneses of plastid genes. These studies have contributed greatly to our understanding of the physiology and biochemistry of biogenergetic processes inside the plastid compartment. Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to lack of pollen transmission. This review describes the generation of plants with transgenic plastids, summarizes our current understanding of the transformation process and highlights selected applications of transplastomic technologies in basic and applied research.     

Synthetic biology in plastids

Plastids (chloroplasts) harbor a small gene‐dense genome that is amenable to genetic manipulation by transformation. During 1 billion years of evolution from the cyanobacterial endosymbiont to present‐day chloroplasts, the plastid genome has undergone a dramatic size reduction, mainly as a result of gene losses and the large‐scale transfer of genes to the nuclear genome. Thus the plastid genome can be regarded as a naturally evolved miniature genome, the gradual size reduction and compaction of which has provided a blueprint for the design of minimum genomes. Furthermore, because of the largely prokaryotic genome structure and gene expression machinery, the high transgene expression levels attainable in transgenic chloroplasts and the very low production costs in plant systems, the chloroplast lends itself to synthetic biology applications that are directed towards the efficient synthesis of green chemicals, biopharmaceuticals and other metabolites of commercial interest. This review describes recent progress with the engineering of plastid genomes with large constructs of foreign or synthetic DNA, and highlights the potential of the chloroplast as a model system in bottom‐up and top‐down synthetic biology approaches.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

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.     

Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants

The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.     

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of 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, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. 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. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.     

Multiple pathways for Cre/lox-mediated recombination in plastids

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.     

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.     

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.     

Genetic engineering of the chloroplast

Transformation of the plastid genome has a number of inherent advantages for the engineering of gene expression in plants. These advantages include: 10-50 times higher transgene expression levels; the absence of gene silencing and position effect variation; the ability to express polycistronic messages from a single promoter; uniparental plastid gene inheritance in most crop plants that prevents pollen transmission of foreign DNA; integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control; and sequestration of foreign proteins in the organelle which prevents adverse interactions with the cytoplasmic environment. It is now 12 years since the first conclusive demonstration of stable introduction of cloned DNA into the Chlamydomonas chloroplast by the Boynton and Gillham laboratory, and 10 years since the laboratory of Pal Maliga successfully extended these approaches to tobacco. Since then, technical developments in plastid transformation and advances in our understanding of the rules of plastid gene expression have facilitated tremendous progress towards the goal of establishing the chloroplast as a feasible platform for genetic modification of plants.     

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.     

Advances in chloroplast engineering

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

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

Breakthrough in chloroplast genetic engineering of agronomically important crops

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development.     

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.     

Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.     

Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid

Transgenic techniques are used to enhance and improve crop production, and their application to the production of chemical resources in plants has been under investigation. To achieve this latter goal, multiple-gene transformation is required to improve or change plant metabolic pathways; when accomplished by plant nuclear transformation, however, this procedure is costly and time consuming. We succeeded in the metabolic engineering of the tobacco plant by introducing multiple genes within a bacteria-like operon into a plastid genome. A tobacco plastid was transformed with a polycistron consisting of the spectinomycin resistance gene and three bacterial genes for the biosynthesis of the biodegradable polyester, poly[(R)-3-hydroxybutyrate] (PHB), after modification of their ribosome binding sites. DNA and RNA analysis confirmed the insertion of the introduced genes into the plastid genome and their polycistronic expression. As the result, the transplastomic tobacco accumulated PHB in its leaves. The introduced genes and the PHB productivity were maternally inherited, avoiding genetic spread by pollen diffusion, and were maintained stably in the seed progeny. Despite the low PHB productivity, this report demonstrates the feasibility of transplastomic technology for metabolic engineering. This "phyto-fermentation" system can be applied to plant production of various chemical commodities and pharmaceuticals.     

植物质体转化途径及其影响因素

植物质体转化开辟了除核基因组之外的另一条遗传转化途径,10几年来取得了很大的进展。但在更多的植物上实现质体转化仍然存在各种困难。本总结了近年来质体转化在各种植物上的研究进展,并针对影响质体转化成功的因素,从组织培养、转化载体和标记基因等几个角度进行了分析。