**3. Making better crops through chloroplast engineering**

with balanced dose of micronutrients. Gene revolution is the only hope for second green revolution to attain these ideal crop cultivars [2]. 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 [3]. 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.

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 [4]. 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 [5]; and production of biopharmaceuticals, vaccine antigens, industrial enzymes, biomaterials, and biofuel [6]. 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 [7]. 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 [8]. 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 [9].

Plastid transformation was first established in unicellular green algae (*Chlamydomonas reinhardtii*) followed by model tobacco plant. It has now been well established in economically valuable crops (rice, brassica, cotton, carrot, spinach, lettuce, etc.) and even in woody plant like popular. Small circular plastid genome (plastome) facilitates targeted engineering, which has been exploited not only for basic research but also for the applied research [10]. Most of the genes present in plastome have been characterized through functional genomics. Organellar transcription and translation have been thoroughly elucidated to understand transcriptional and translational machinery of the plastids. Even the proteins involved in cross-talk between chloroplast and nucleus have been worked out. Further, plastid transformation is the most ideal

**2. Chloroplast biotechnology—an overview**

62 Transgenic Crops - Emerging Trends and Future Perspectives

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 [11] attempted to express complete RuBisCO protein in tobacco from *Synechococcus elongatus* by disrupting the host native enzyme. CO2 fixation rate and carboxylase activity of the RuBisCO were increased, especially at higher concentrations of CO<sup>2</sup> . Hence, photosynthetic performance can be improved by introducing more competent complete photosynthetic system into a plant. Raising concentration of CO<sup>2</sup> in plastids is another possibility strategy, to improve photosynthetic carbon fixation and crop productivity. Cyanobacterial bicarbonate transporter was expressed in tobacco plastid genome, but any considerable improvement in photosynthetic performance was not observed. Expression of fructose-1,6-sedoheptulose-1,7-bisphosphatase in lettuce and tobacco chloroplasts appeared to increase productivity of engineered plants. Likewise, chloroplast-encoded chlB gene from *Pinus thunbergii* was found to promote root growth and early chlorophyll pigment development in tobacco [12]. Hence, research is in progress to engineer C3 plants to C4 by manipulating RuBisCO large subunit and photorespiratory pathway for enhanced biomass production [13].

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 [14]. 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 [15]. Expression of agglutinin gene (pta) in leaf chloroplasts resulted in broad spectrum resistance against lepidopteran insects, aphids, and viral and bacterial pathogens [16]. A gene stack comprising CeCPI (sporamin, taro cystatin) gene from sweet potato and chitinase from *Paecilomyces javanicus* was introduced into tobacco, and resultant plants showed resistance not only against various pests and diseases but also against salinity, osmotic stress, and oxidative stress [17]. Expression of osmoprotectant (yeast trehalose phosphate synthase) in tobacco plastids resulted in 20-fold higher trehalose accumulation; as a result, plants were tolerant to drought and osmotic stress [18]. The overexpression of *mdar* transgene in tobacco plastids and the fusion of such chloroplasts to Petunia cells were suggested to possibly protect the plants against oxidative stress. Oxidative stress tolerance was also enhanced in transplastomic tobacco plants expressing flavodoxin (fld) from cyanobacteria. Transplastomic plants overexpressing panD not only appeared to produce 30–40% higher biomass but also appeared to be more tolerant to increased heat stress. Similarly, expression of arabitol dehydrogenase (*ArDH)* in tobacco chloroplasts enabled them to survive even at 400 mM NaCl [19]. Chilling tolerance as well as growth was observed to be increased in tropical forage *Stylosanthes guianensis* expressing chloroplast protein 12 [20]. Recently, plastid transformation has been reported in a valuable vegetable *Momordica charantia* [21], marine microalga *Nannochloropsis oceanica* [22], and *Cyanidioschyzon merolae* [23]*,* a red alga having ability to survive in high sulfur acidic hot spring environments. This may open new horizons in understanding stress adaptability and role of transplastomic technology in developing stress-tolerant plants.

calcium uptake machinery is present on the membranes; so, the thylakoid membrane may be the major sequester of Ca2+ in chloroplasts [28]. It was further elucidated that activity of these transporters is regulated by light or photosynthesis, so chloroplasts take up calcium during the day and store it in the lumen or some identified sequestering sites. During the night, Ca2+ is released from the store houses for long-lasting, dark-induced Ca2+ signals; hence, sensing of photoperiod and light/dark transition seems to be regulated by Ca2+ signaling. In addition to light, Ca2+ signaling is also influenced by other abiotic stimuli (salinity, cold) and hence plays

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 *Arabidopsis* are AtACA1 and AtHMA1. AtACA1 is an autoinhibited Ca2+ transporter, which is predicted to be targeted to the chloroplasts. It is specifically expressed in the root and is then localized to endoplasmic reticulum or tonoplast. AtHMA1 is a heavy metal P-type ATPase in the chloroplast envelope and plays an important role in calcium transportation [29]. Recently, another transporter AtGLR3.4 (glutamate receptor) has been explored to form Ca2+ permeable nonselective cation channels and is localized in the chloroplasts [30]. In addition, two MscS homologs, localized in the chloroplast, have also been evaluated to be essential for plastidic osmoregulation [31]. Since these transporters play a

plays a significant role in Ca2+ uptake via thylakoid proton gradient. Pea thylakoid protein (PPF1) is another candidate calcium transporter at thylakoid membrane, which has been found to enhance Ca2+ currents when tested in human cultured cells. These findings demonstrate that Ca2+-binding protein and Ca2+ transporters play a significant role in immune signal transduction pathway. Anyhow, most of the genes involved in these pathways are

Plastids are known to get evolved from primitive cyanobacteria through a process known as endosymbiosis [32]. 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 [33]. *In vivo* genes of plastid may be present as a linear molecule or a complex branched form, and many copies of plastid genome can be harbor in each organelle. Size of plastid genomes varies from <100 to >1000 kbp (kilobase pair). The region of small single copy (SSC) and large single copy (LSC) are separated by two inverted repeats (IRs) present in the plastid genome (**Figure 1**). The thymine and adenine residues are often rich in plastid genome; a reductive evolution is also seen in those of mitochondrial genomes and bacterial endosymbionts [34]. Noncoding DNAs are abundantly present in some plastid genomes, while the others have self-splicing introns. The genome of some dinoflagellates spreads across a sea of minicircles; recently, multiple linear chromosomes formed a hairpin structure, which

antiporter also

Technical Advances in Chloroplast Biotechnology http://dx.doi.org/10.5772/intechopen.81240 65

key role in the sequestration of calcium ions into the thylakoid lumen, Ca2+/H+

**5. Advances in plastid functional genomics**

have been found in the plastid genome of certain green algae [35].

some crucial role in stress tolerance as well.

still to be elucidated.

#### **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 [24], have their own Ca2+ binding proteins, and Ca2+ sensors, which are expected to play a significant role in Ca2+ signaling within the plant cell [25]. 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 [26]. The concentration of free calcium was found to be very low when determined by targeting apoaequorin to the stroma of tobacco chloroplasts [27]. 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 *Arabidopsis thaliana* revealed out that active calcium uptake machinery is present on the membranes; so, the thylakoid membrane may be the major sequester of Ca2+ in chloroplasts [28]. It was further elucidated that activity of these transporters is regulated by light or photosynthesis, so chloroplasts take up calcium during the day and store it in the lumen or some identified sequestering sites. During the night, Ca2+ is released from the store houses for long-lasting, dark-induced Ca2+ signals; hence, sensing of photoperiod and light/dark transition seems to be regulated by Ca2+ signaling. In addition to light, Ca2+ signaling is also influenced by other abiotic stimuli (salinity, cold) and hence plays some crucial role in stress tolerance as well.

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 *Arabidopsis* are AtACA1 and AtHMA1. AtACA1 is an autoinhibited Ca2+ transporter, which is predicted to be targeted to the chloroplasts. It is specifically expressed in the root and is then localized to endoplasmic reticulum or tonoplast. AtHMA1 is a heavy metal P-type ATPase in the chloroplast envelope and plays an important role in calcium transportation [29]. Recently, another transporter AtGLR3.4 (glutamate receptor) has been explored to form Ca2+ permeable nonselective cation channels and is localized in the chloroplasts [30]. In addition, two MscS homologs, localized in the chloroplast, have also been evaluated to be essential for plastidic osmoregulation [31]. Since these transporters play a key role in the sequestration of calcium ions into the thylakoid lumen, Ca2+/H+ antiporter also plays a significant role in Ca2+ uptake via thylakoid proton gradient. Pea thylakoid protein (PPF1) is another candidate calcium transporter at thylakoid membrane, which has been found to enhance Ca2+ currents when tested in human cultured cells. These findings demonstrate that Ca2+-binding protein and Ca2+ transporters play a significant role in immune signal transduction pathway. Anyhow, most of the genes involved in these pathways are still to be elucidated.
