Chloroplasts are highly organized cellular organelles after master organelle nucleus. They not only play a central role in photosynthesis but are also involved in several crucial cellular activities. Advancements in molecular biology and transgenic technology have further groomed importance of the organelle, and they are the most ideal ones for the expression of transgene. No doubt, limitations are there, but still research is advancing to resolve those. Certain valuable traits have been engineered for improved agronomic performance of crop plants. Industrial enzymes and therapeutic proteins have been expressed using plastid transformation system. Synthetic biology has been explored to play a key role in engineering metabolic pathways. Further, producing dsRNA in a plant’s chloroplast rather than in its cellular cytoplasm is more effective way to address desired traits. In this chapter, we highlight technological advancements in chloroplast biotechnology and its implication to develop biosafe engineered plants.
- chloroplast biotechnology
- value-added crops
- trouble-rescue organelles
- plastid functional genomics
Food security is a long-lasting challenge for the growing world and is becoming more alarming in the developing countries where one out of every nine people is malnourished. So-called processing (polishing, milling, and pearling) of the cereals makes them even poorer in micronutrients . Climate change is another challenge that poses continuous stress on the crop productivity. Sharply decreasing arable soils and use of heavy inputs to get high crop yield are further deteriorating our environment and quality of available food. All this demands availability of improved crop cultivars having ability to perform in the changing climate scenario and even with balanced dose of micronutrients. Gene revolution is the only hope for second green revolution to attain these ideal crop cultivars . Since the commercialization of transgenic crops in 1994, the area under GM crops is sharply increasing and has now increased to 180 million hectares. This includes crops for improved agronomic traits (herbicide tolerance and insect resistance, salinity and drought tolerance, and efficient nutrient utilization), enhanced level of micronutrients, and for the expression of therapeutic proteins and industrially important enzymes. At the same time, emotional or obsolete arguments are there to oppose the use of GM (genetically modified) products. Opponents of GM crops have straightforwardly rejected genetically modified products and have produced questionable scientific data to ban their commercialization. Plastid biotechnology has emerged as a competent field of research having potential to address all of the questions raised by the opponents of GM crops . This chapter highlights significance of plastid transgenic technology to develop valuable crop plants. Further, technological advancements have been discussed to get an update about the recent research to resolve existing bottlenecks in the development and commercialization of transplastomic plants.
2. Chloroplast biotechnology—an overview
Transgenic technology is the technology of the day to develop crop plants with desired traits but crucial traits need to be engineered through plastid genome instead of nuclear genome . It is an amazing organelle where more than 120 genes from various sources have been integrated and expressed. This organellar genome has well been explored for a wide variety of applications including crops with elevated level of resistance against biotic (insects, bacterial, viral, and fungal diseases) and abiotic stresses (salinity, drought, and cold); phytoremediation of toxic metals, cytoplasmic male sterility ; and production of biopharmaceuticals, vaccine antigens, industrial enzymes, biomaterials, and biofuel . Hyperexpression of recombinant protein in plant expression system is only possible through plastid transformation. The high ploidy number of the plastid genome results in higher level of protein expression, and up to 70% total soluble protein is reported to be produced in tobacco . Moreover, hyperexpression of therapeutic proteins and vaccine antigens in chloroplasts (leaves), leucoplasts (roots), or chromoplasts (fruits) makes it ideal organelle for the oral delivery of vaccine antigens against tetanus, cholera, anthrax, canine parvovirus, and plague . Other salient advantages include possibility of multigene engineering, absence of gene silencing, position effect, epigenetic, complete absence of pleiotropic effects due to subcellular compartmentalization, and transgene containment due to maternal inheritance of plastids in most of the crops .
Plastid transformation was first established in unicellular green algae (
3. Making better crops through chloroplast engineering
It is predicted that sharply increasing population necessitates an increase in crop yield at 30% per annum. In this scenario, chloroplast biotechnology is the most ideal approach to develop crop plants with improved photosynthetic performance, enhanced nutritional value, improved agronomic traits, and producing valuable fatty acids. Plastid transformation was first established in flowering plants almost 30 years ago. Though it has been extended to other crop plants, most of the studies have been conducted in tobacco, which is nonfood nonfeed crop. This demands further efforts by the scientific community to engineer plastid genome of valuable crop plants for desired traits, leading to increased quality and quantity of food.
Most of the efforts to increase crop productivity had been made to improve photosynthetic performance of the plants. RuBisCO (the core enzyme of photosynthesis), large subunit, is encoded by chloroplasts, whereas small subunit is encoded by nuclease, which is then imported to chloroplast. Efforts have been made to engineer RuBisCO large subunit, small subunit, or both. Lin  attempted to express complete RuBisCO protein in tobacco from
Insect resistant crops had successfully been grown in the field since 1994. Resistance development against Bt crops is an emerging concern, which needs to be addressed through high-dose strategy and gene pyramiding. Another possibility to develop insect-resistant transplastomic plants is the upregulation of their pathogen defense mechanisms. Expression of β-glucosidase in tobacco plastome showed not only growth of the plants but also more resistance against insect pests . A novel non-Bt-type insect resistance strategy has been evaluated by expressing dsRNA, targeting an essential insect gene in transplastomic plants. Disruption of target gene by RNA interference resulted in 100% mortality in adult beetles and in the larvae within 5 days of feeding . Expression of agglutinin gene (pta) in leaf chloroplasts resulted in broad spectrum resistance against lepidopteran insects, aphids, and viral and bacterial pathogens . A gene stack comprising CeCPI (sporamin, taro cystatin) gene from sweet potato and chitinase from
4. Chloroplasts as trouble-rescue organelles
Chloroplasts not only are the central hubs for photosynthesis but also have evolved as fundamental trouble-rescue organelles. Recent studies have revealed that chloroplasts play a key role in switching plants from vegetative mode to defense mode. In addition to intraorganellar functions, they also play crucial role in the regulation of extraorganellar processes such as plant stress response, apoptosis, and immunity. Both of the cellular organelles (chloroplast and mitochondria) evoke their own particular Ca2+ signals , have their own Ca2+ binding proteins, and Ca2+ sensors, which are expected to play a significant role in Ca2+ signaling within the plant cell . As a result, they have capacity to sequester and serve as sink for Ca2+, which plays a key role in physiological and environmental responses of eukaryotic cells.
Chloroplasts are important intracellular calcium (Ca2+) stores and may accumulate up to 15 mM or even higher. Most of the plastidic Ca2+ resides within the stroma or thylakoid membranes through interaction with calcium-binding proteins . The concentration of free calcium was found to be very low when determined by targeting apoaequorin to the stroma of tobacco chloroplasts . Hence, stroma is not the major sequester of Ca2+ in chloroplasts. This helped to elucidate that chloroplasts have their own active transporters on the envelope membranes, which help them to accumulate high concentrations of Ca2+ within the thylakoid membranes or some other unidentified Ca2+ stores. Identification of CAS (high capacity Ca2+-binding protein) in the thylakoid membranes of
An active Ca2+ uptake machinery is present in chloroplast, which is regulated by transporters. Much research has not been conducted on these transporters; as a result, only few are known, whereas others are still to be elucidated. Two potential membrane transporters (Ca2+-ATPase) in
5. Advances in plastid functional genomics
Plastids are known to get evolved from primitive cyanobacteria through a process known as endosymbiosis . Although plastid genomes are much smaller as compared to their cyanobacterial progenitors, similarities in gene sequence as well as genome topology are evident. Just like cyanobacterial genome, plastid genomes are tightly packed with genes as a circular molecule .
A huge portion of the cyanobacterium derivative genes required for plastid function now exist in the nucleus, having transferred through a process known as endosymbiotic gene transfer (EGT). Subsequently, most of the plastome proteins are introduced posttranslationally. Nevertheless, genomes of plastid normally encode some of their own processing machinery, including ribosomal proteins, ribosomal RNAs, bacterial RNAs polymerase, and tRNAs—however, land plants also have nuclear-encoded plastid RNA polymerases. Remarkably, genome of plastid also encodes many photosynthesis components, such as proteins of photosystem I and II (e.g.,
6. Role of synthetic biology in engineering plastid metabolic pathways
During past two decades, the synthetic biology approach has brought about several remarkable accomplishments regarding engineering of biological systems particularly microbes and yeast. However, such promising attributes of synthetic biology have not been explored for plastid genome engineering except few striking instances . The future of the recombinant DNA technology is linked with advancement and implications of synthetic biology because development of novel biological systems is dependent on this area of research. Like traditional disciplines of engineering, synthetic biologists also use abstraction, standardization, and decoupling to design more efficient biological systems . So, to design an organism of choice synthetic biology is of fundamental importance. Unique advantages of plastid transformation technology regarding metabolic pathway engineering make it more important than nuclear transformation technology. Hence, coalescing synthetic biology with plastid genome engineering can be more fruitful and valuable for the production of economical recombinant proteins . However, understanding plastid genomics is equally important in order to harvest potential benefits of synthetic biology. The anterograde and retrograde signaling (nucleus to plastid and vice versa) of plastid proteins revealed that most of the protein complexes were chimeric and contained both plastid encode subunits as well as nucleus encoded subunits. Further, nucleus encoded proteins follow eukaryotic mode of expression, whereas plastid encoded proteins follow prokaryotic mode of expression, though plastid genome is quite smaller in size than nuclear genome (less than 10%) . Since efforts have been made to develop synthetic plastid genome of minimum size for efficient transplantation into a cell without plastids or to replace native plastid genome with engineered operons coding for valuable proteins. This requires information about the most essential genes involved in the stability and integrity of the plastome. Owing to high cost of synthetic DNA, initially it was used only for the optimization of codon usage of transgene, but now it is affordable to synthesize complete vector or genome. This not only avoids intensive cloning work but also facilitates synthesis of multiple genes with desired regulatory sequences. Use of synthetic expression elements has helped to get appropriate expression of transgene in nongreen plastids including tubers and fruits . Chloroplast being metabolic center of the cell is the most attractive organelle whose metabolic pathways need to be engineered. Further, it has ability to stack multiple synthetic operons. Major limitation in this context is size of the transgene as engineering metabolic pathways require engineering of the multiple genes involved in that particular pathway [42, 43]. The identification of intercistronic expression elements (minimum sequence elements involved in the proper processing of polycistronic transcript into monocistronic) has helped to devise workable synthetic operons for the expression of multiple proteins involved in the biosynthesis of vitamin E , artemisinic acid , carotenoids , and dhurrin  or other metabolic pathways . Likewise, synthetic operons can be helpful for the transformation of C3 photosynthetic pathway into C4, engineering of nitrogen fixation pathway, or molecular farming for the production of industrial enzymes and therapeutics .
7. Regulation of RNA editing in chloroplasts
An important process of gene regulation is RNA editing. This occurs at posttranscriptional level through nucleotide modification for many functional genes. RNA editing restores the conserved amino acid residues for functional proteins in plants. Changes in RNA sequence of functional gene occurs during RNA editing, through the molecular mechanisms . Cytidine-to-uridine editing and adenosine-to-inosine editing are two types of RNA editing identified in
In chloroplast gene expression system, RNA editing is an important posttranscriptional modification. The use of pentatricopeptide repeat (PPR) protein family for RNA editing in chloroplast has been reported . Mostly genes in chloroplast are cotranscribed and arranged in clusters. To control gene expression, posttranscriptional RNA editing is an essential step, and this step is also required for gene function . It has been studied that C-to-U editing is the major type of RNA editing in chloroplasts. In chloroplast, etioplast, and amyloplast of maize, expression of almost 15 different genes has been affected by 27 C-to-U RNA editing sites. In chloroplast, RNA editing plays an important role to correct harmful mutations instead of producing protein diversity. Genomic DNA sequence is not changed by C-to-U editing because this editing changes the nucleotide sequence only within RNA molecule. RNA polymerase is used to produce RNA editing . Insertion, deletion, and base substitution are events of RNA editing. That is why RNA editing can reverse harmful genomic mutations in consistent RNA transcript. In chloroplast, different sites are edited by C-to-U RNA editing enzymes as well . Around 126 C-to-U editing events and 11 U-to-C editing events were identified in the chloroplast DNA of moth orchid (
8. Conclusions and future directions
Chloroplasts are the most important solar-energy-capturing natural systems on earth. They not only capture it but also convert it into a form useful for all living organism on earth. Molecular oxygen is liberated as a by-product, which is a vital source for respiration of all aerobic organisms. Chloroplasts are believed to be evolved from prokaryotic ancestors through a process known as endosymbiosis. Chloroplast contains circular genome having compactly arranged genes, which are involved in not only photosynthesis but also many other vital biological processes. Keeping in view its utmost physiological importance, plant as well as algal plastome has been engineered for a number of agronomic as well as pharmaceutical traits [63, 64]. Advancements in molecular biology and transgenic technology have further groomed importance of the organelle, and they are the most ideal ones for the expression of transgene. Resolving current limitations including vector design, gene regulation control and DNA delivery may further improve this important field of biotechnology . Synthetic biology is being explored in this regard, which is expected to play a major role in enhancing contribution of chloroplasts not only for sustainable food production but also for other important molecules in future.