**7. Regulation of RNA editing in chloroplasts**

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].

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

Though it is the beginning of plastid synthetic biology, advancements are being made to develop the essential tools regarding transgene expression control in chloroplast genome [37]. Currently, most recombinant expression in the plastids involves single-gene constructs

**6. Role of synthetic biology in engineering plastid metabolic** 

**pathways**

two inverted repeats (IRa and IRb).

66 Transgenic Crops - Emerging Trends and Future Perspectives

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 in *Arabidopsis thaliana* [51]. RNA editing is a rare process where RNA polymerase is involved in insertion, deletion, and base substitution of nucleotide within the transcript [52–55]. Many studies reported the evidence of RNA editing in tRNA, rRNA, and mRNA. However, RNA editing has also been reported in noncoding RNA, like microRNAs of eukaryotes. The RNA editing occurs in all DNA-containing organelles like nucleus, mitochondria, and plastids. In nucleus, chloroplast and mitochondria RNA editing occurs during the process of transcription and posttranscriptional modifications [56, 57]. Caseinolytic protease complex component (CLPC1) plays a crucial role in RNA homeostasis [58]. Anyhow, discrete changes in RNA before its translation into protein occur by RNA editing. Besides this, RNA editing is also a vibrant mechanism to produce functional and molecular diversity [59].

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 [65]. 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

Technical Advances in Chloroplast Biotechnology http://dx.doi.org/10.5772/intechopen.81240 69

Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture,

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[2] Solymosi K, Bertrand M. Soil metals, chloroplasts, and secure crop production: A review. Agronomy for Sustainable Development. 2012;**32**:245-272. DOI: 10.1007/ s13593-011-0019-z

[3] Khan MS. Plastid genome engineering in plants: Present status and future trends.

[4] Adem M, Beyene D, Feyissa T. Recent achievements obtained by chloroplast transforma-

[5] Khan MS. Plant biology: Engineered male sterility. Nature. 2005;**436**:783-785. DOI: 10.1038/

[6] Richter LV, Yang H, Yazdani M, Hanson MR, Ahner BA. A downstream box fusion allows stable accumulation of a bacterial cellulase in *Chlamydomonas reinhardtii* chloro-

[7] Bock R. Engineering plastid genomes: Methods, tools, and applications in basic research and biotechnology. Annual Review of Plant Biology. 2015;**66**:31-33. DOI: 10.1146/

[8] Michoux F, Ahmad N, Hennig A, Nixon PJ, Warzecha H. Production of leafy biomass using temporary immersion bioreactors: An alternative platform to express proteins in transplastomic plants with drastic phenotypes. Planta. 2013;**237**:903-908. DOI: 10.1007/

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production but also for other important molecules in future.

\*Address all correspondence to: drmustafa8@gmail.com

Molecular Plant Breeding. 2012;**3**:91-102

plasts. Biotechnology for Biofuels. 2018;**11**:133

tion. Plant Methods. 2017;**13**:30

annurev-arplant-050213-040212

s00425-012-1829-1

Muhammad Sarwar Khan, Ghulam Mustafa\* and Faiz Ahmad Joyia

**Author details**

Faisalabad, Pakistan

009-0046-6

436783a

**References**

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 [51]. 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 [52]. 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 [60]. 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 [61]. Around 126 C-to-U editing events and 11 U-to-C editing events were identified in the chloroplast DNA of moth orchid (*P. aphrodite* subsp. Formosana). In leaf tissues, 110 editing events and in floral tissue, 106 editing events were identified. In non-protein-coding RNA such as introns, tRNA, and regulatory sequences, RNA editing occurred [62]. Besides C-to-U editing, which is mostly reported in chloroplast of plants, adenosine-to-inosine editing in plastid tRNA of *Arabidopsis thaliana* has also been characterized. Adenosine-to-inosine editing was recognized in the anticodon of the plastid tRNA-Arg (ACG). AtTadA gene expression is involved in adenosine-to-inosine editing in the chloroplast [51].

### **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 [65]. 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.
