**5. Advances in plastid functional genomics**

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.

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

**4. Chloroplasts as trouble-rescue organelles**

64 Transgenic Crops - Emerging Trends and Future Perspectives

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

created using conventional restriction enzyme-based cloning approaches. This limits the rate at which new transplastomic lines can be produced and tested, and in particular, how many different permutations of constructs (different promoters, coding variants, regulatory elements, etc.) can be evaluated. Currently, various synthetic biology principles are being applied to plastome engineering with the adoption of assembly standards such as Golden Gate and the creation of libraries of validated DNA parts that allow rapid one-step assembly of all the parts [38–40]. More ambitious design strategies involving extensive redesign of the plastome *in silico* are expected to be optimized and validated. Some of the foreseen strategies include removal of large tracts of nonessential DNA [41], refactoration of numerous essential endogenous genes into functional clusters [42], and synchronized engineering of multiple transgenes into different loci. The assembly and delivery of such synthetic genomes is technically feasible, as shown by O'Neill et al. [37], who demonstrated that the entire *C. reinhardtii* plastome could be assembled in yeast and transformed into *C. reinhardtii* by microparticle bombardment. Plastidic intercistronic expression elements (IEEs) can be used for the expression of synthetic operons [43]. The challenge is to develop selection strategies that allow the clean replacement of the endogenous plastome with the synthetic version without undesirable recombination events between the two, resulting in the creation of chimeric plastomes. Another challenge is to improve the product yield significantly through the use of synthetic cis elements to drive expression. Currently, the promoter and 5′ UTR used to express transgenes are derived from endogenous photosynthetic genes. In some cases, expression levels can be improved by using the stronger promoter from the gene for the 16S ribosomal RNA fused to the 5′ UTR of a photosynthetic gene [44]. However, more often it is the performance of the 5′ UTR that is the bottleneck [45], with the efficiency of translation constrained by either the same feedback regulation that prevents the overaccumulation of individual photosynthetic subunits in the absence of their assembly partners (so-called "control by epistasy of synthesis") or by competition with the corresponding endogenous gene transcript for transacting factors that are required for transcript stability or translation but are present in limiting concentration in the chloroplast [46]. The strategies to overcome this involve either replacement of the 5′ UTR of the endogenous gene with that from another photosynthetic gene [47] or, more elegantly, the development of synthetic variants of the 5′ UTR that are no longer subject to these limitations and therefore enable improved expression of the transgene [48]. Further studies into the design of synthetic promoters and UTRs, combined with improved knowledge of codon optimization rules, will advance the ability to engineer plastid metabolic pathways for customized functionalities and production efficiencies of commercial scale [49].

Technical Advances in Chloroplast Biotechnology http://dx.doi.org/10.5772/intechopen.81240 67

**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 [50]. Cytidineto-uridine editing and adenosine-to-inosine editing are two types of RNA editing identified

**Figure 1.** Circular map of chloroplast genome showing one large single copy (LSC), one small single copy (SSC), and two inverted repeats (IRa and IRb).

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., *psbA* gene of photosystem II coding for the D1 unit) as well as cytochrome *b6f*, which facilitates electron transfer between both photosystems I and II [36].
