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ABSTRACT

The genome of the plastids is tetrapartite i.e. having a small single-copy region, large single-copy region and inverted-repeat regions in between them, but the mitochondrial genome which is multipartite, vary to a large extent with regards to size and organization in species. Owing to its multipartite nature and high recombination activities, mitochondria can evolve much faster than chloroplast genome. But the unstable of mitochondrial genome and lack of selectable markers are some of its bottlenecks in transformation of the mitochondrial genome in higher plants. In spite of many proof-of-concept usage, commercialization of transplastomic plants have not been successfully done till date. To modify nuclear genomes, sequence-specific nuclease technologies are extensively used, but they have not been applied for organellar genome editing as the plastid genome is efficiently edited by homologous recombination system. Unlike transplastomics, genetic transformation of higher plant mitochondrial genome has been attempted but has not been successful till date.

Keywords: Plastid, mitochondria, genome, transplastomic plants, crop improvement

The plant cells comprise of three different types of DNA i.e., nuclear DNA, chloroplast DNA and mitochondrial DNA. The plants developed by transformation of plastids by use of biotechnological techniques is called as transplastomic plants. Genetically engineered plastid DNA can help improve the crop productivity, quality traits, and resistance to various stresses both biotic and abiotic and produce recombinant proteins. Till date transplastomic has been limited to labs. Homologous recombination system has been found to be effective plastid editing technology (Li et al., 2021). Successful mitochondrial genetic transformants have not been produced owing to its unstable genome. Although, attempts are taken to study the modified genes and their transcripts to know the functionality of the genes. In spite of many proof-of-concept usage, commercialization of transplastomic plants have not been successfully done till date (Li et al., 2021).

To modify nuclear genomes, sequence-specific nuclease technologies are extensively used, but they have not been applied for organellar genome editing as the plastid genome is efficiently edited by homologous recombination system. Unlike transplastomics, genetic transformation of higher plant mitochondrial genome has been attempted but has not been successful till date.  (Li et al., 2021). Though, bit-by-bit progress has been made in altering mitochondrial genes and their transcripts, accordingly enabling the study of their functions. By 2060, the population of the world is estimated to have crossed 10 billion. To feed the ever-increasing population, the system of food production has to enhance by increasing the produce and reducing the inputs requirements simultaneously. In the era genetic engineering, the time and labour requirements have drastically gone done and productions of transformants have become far more rapid and easy. The plant cells comprise of three different types of DNA i.e., nuclear DNA, Chloroplast DNA and mitochondrial DNA. These DNA have their own functions and they replicate independently of each other (Li et al., 2021). This article provides an outline on the works recently done to achieve transformation in organelle, its limitations and future prospects.

 Plastid genome

In higher eukaryotic plants plastid genome is conserved, about 150 kb size, consisting of about 130 genes. They are divided into the following parts i.e., SSC- small single-copy region, LSC - large single-copy region and IRR- inverted-repeat regions (Two copies). Formation of transgenic plants by transforming plastids, for the first time, it was successfully achieved in Chlamydomonas reinhardtii (an algae) and among higher plants, it was achieved in tobacco (Nicotiana tabacum).  Now it has been successfully implemented in over 20 flowering species. The size in base pairs (bp) of some plant chloroplast genome is indexed in Figure 2. Now, highly competent transplastomic plants model of Arabidopsis has been achieved (Li et al., 2021). Unique advantages of transformed plastid genomes over usual nuclear genetic engineering are precise introduction of transgene by homologous recombination method, expression of foreign gene to unusually high levels, multistacked transgenes by use of synthetic operons and nonappearance of epigenetic effects. The sizes in base pairs with accession number of some plant chloroplast genome are indexed in Figure 1.

 Mitochondrial genome

Plants have vast and complicated mitochondrial genomes in comparison to mammals. Size (in base pairs) of some plant mitochondrial genome is indexed in Figure 2. Almost all mitochondrial animal genomes are around 16.5 kbp long, but the mitochondrial genomes of the plants span from 200 to 2,000 kbp. This is fascinating to suppose that current mitochondria’s come from a common ancestor of alpha-proteobacter. Plant mitochondrial genomes do not contain considerably more genes than their animal counterparts, despite their size. In the plant mitochondrial genomes, most extra DNA comprises of huge introns, repeats and noncoding sequences. Moreover, plant mtDNA does not exist as big circular DNA molecules but mostly as a collection of linear DNA with smaller circular and branching combinations. Studies of these extremely fragmented genomes strongly suggest that recombination is the major mechanism for the replication of mtDNA plant. Its size is variable from 208 kb to 11 Mb and hence is not conserved as that of chloroplast DNA. But the number of genes in the mitochondrial genome is conserved and it contains about 60 genes in almost all land dwelling plants. Maximum part of its genome are introns. However, unswerving approaches by using biolistic device for the alteration of mitochondria has recently been used only for yeast and green algae and no positive transformation of mitochondria in the higher plant has been stated till date. Although, the mitochondrial genomes of higher plants are not transformable, modifications have been achieved in Arabidopsis and rice by use of TALENs.

  Plastid genome editing for crop improvement

 

(i)      Enriched photosynthesis in crop plants

To feed the ever-exploding population, agricultural productivity has to increase significantly and this can be achieved by increasing the photosynthetic ability of crop plants. In the Calvin cycle, Rubisco (Ribulose-1, 5-bisphosphate carboxylase/oxygenase) is the chief enzyme which is responsible for assimilating carbon dioxide. Rubisco is the most abundant plant protein in nature. It comprises of 8 large catalytic subunits (rbcL) encoded by genome of the plastid and 8 small subunits (rbcS) that are encoded nuclear genome. For quite some time, Rubisco been viewed as a restricting factor for photosynthesis and crop yield due to its poor capacity to differentiate between carbon-dioxide & oxygen and its slow catalytic rate.  One methodology is to migrate a rbcS gene back into its pre-endosymbiotic area inside the chloroplast genome to gather functional Rubisco in chloroplasts (Li et al., 2021). For example, all endogenous rbcS genes are silencing to develop to bRrDs tobacco line. The tobacco rbcL genes were substituted with the Rhodospirillum rubrum rbcM gene, which encodes the L2 form of Rubisco that does not assemble with the small subunit. By this experiment, the maximum amount of Rubisco produced touched only 50% of the wild types’ levels. This work allows the designing of various Rubisco subunit assemblies and adds to endeavours to design more prominent quantities of Rubisco. Another procedure to further develop photosynthesis of the plant is the incorporation of a carbon dioxide concentrating instrument into transplastomic plants from cyanobacteria to expand their fixation of carbon (Li et al., 2021).

 

(ii)    Metabolic transformation in crop plants

Plastid is a site of large number of biosynthetic pathways which produces a wide range of precursors for many significant secondary metabolites like phytohormones and pigments. One of the greatest attractions of plastid transformation is the likelihood of stacking of transgene in synthetic operons for the engineering of complex biochemical pathways. For example, core artemisinic acid biosynthetic pathway expressed by Transplastomic tobacco led to low level of accumulation of the metabolites. COSTREL (combinatorial supertransformation of transplastomic recipient lines) method was utilised to increase the accumulation of artemisinic acid. COSTREL lines were found to accumulate artemisinic acid by 77 times (Li et al., 2021). This analysis provided evidences for combining plastid and nuclear transformation to enhance product yields from complex biochemical pathways in chloroplasts.

 (iii)  Resistance to insects in crop plants

Expression of Bt (Bacillus thuringiensis) cry1A(c) gene in the plastids of tobaaco has been the first case of successful transformation of plastids for with regard to pest control. Accumulation of large amount of Bt protein (3-5% of TPS) which is insecticidal by its property has been found to be accumulated in the transgenic plastids of tobacco plants. Expression of cry2Aa2 along with 2 open reading frames, is found to accumulate insecticidal protein upto 45% of TSP. Though, high level of expression of cry9Aa2 (10% of TSP) from the tobacco triggered acute retardation of growth of the transplastomic plants. Thus, it was concluded that expression of transgenes needs to be judiciously optimized to a desirable level where it works against the insects and does not hamper the plant growth too (Li et al., 2021). High death rate of two lepidopteran caterpillars, Lymantria dispar and Hyphantria cunea have recently been found to be due to cry1C gene expression from the plastid genomes. High death rate of a leaf-eating beetle, Plagiodera versicolora has been recorded in transplastomic expressing cry3Bb gene. In the transplastomic technology, other than Bt genes, expressing long double-stranded RNA (dsRNA) in plastids to aim an vital insect gene can be developed the novel pest control scheme.

 Mitochondrial genome editing towards molecular breeding in crop plants

Though, much information is available about the mitochondrial genome sequence in higher plants but precise tool to transform mitochondrial genomes has been a limiting factor in its studies (Li et al., 2021). Transformations in mitochondrial genomes have been attempted by using the following technologies:

·   Nuclear transformation via TALEN technology in which genes associated with cytoplasmic male sterility in rice and rapeseed have been knock out precisely by use of mitoTALENs technology.

·   CRISPR/Cas9 machinery along with gRNA has been applied for mitochondrial transformation of mammalian cells. They can also be used to edit mitochondrial RNA transcripts using PPR proteins. This techniques new opportunity for the studies of reverses genetics and has a tremendous potential usage in the field of agriculture.

·   DYW-type PPRs could be used for precise C-to-U modification and to generate or destroy start and stop codons in the mRNA of the organellar gene to regulate its translational activities.

·   CRISPR-Cas13 can also be used as an RNA editing tool provided a accurate gRNA delivery system to the matrix of mitochondrial could be developed.

 Application of RNA editing in crop improvement

RNA editing refers to the base pair changes made after the transcription in the mRNA with respect to their corresponding gene. Three decades ago, RNA editing from C-to-U was discovered in the mitochondria of angiosperms and have recently been reported in chloroplast. In the RNAs of mitochondria 300 to 600 editing sites are present whereas in chloroplast only 20 to 60 sites are present. Almost all the editing sites are present in coding region of the genes except a few which are present in the non-coding introns. Editosome is responsible for the editing of the RNA. It contains multiple protein factors hence, called as multiprotein complex. Thought the RNA editosome is composed of a variety of editing factors, one of the main components is PPR proteins (Li et al., 2021). PPR proteins are composed of 35-aminoacid helix–turn–helix motifs. The PPR present in flowering plants can be divided into two sub families i.e., P and PLS subfamilies (Table 1). The PLS subfamily protein is responsible for in C–U RNA editing in plant organelles (Li et al., 2021). PPR helps recognise specific RNA bases. This interaction makes them liable to bring about modifications in the plastids. The RNA editing site of the plastids is not strictly conserved and has been lost frequently during evolution (Li et al., 2021).

Table 1. PPR classification in flowering plants

 

Sub family type	Specification
The P-type PPRs	Only canonical motifs of 35 amino acids- plays role in RNA end maturation, intron splicing, etc.
The PLS-type PPRs	consists of longer L-type (35–36 amino acids)
Shorter S-type	31–32 amino acids motif variants

 Conclusion

Plastid transformation technology has led to development of transgenes, RNA editing and genome editing in plastids. Transplastomes have been successfully developed in tobacco and Chlamydomonas. Presently available methods of selection and protocols of regeneration are mainly the limitation to the technology. Currently, gene that causes resistance to tobramycin i.e., aminoglycoside acetyltransferase (60)-Ie/ aminoglycoside phosphotransferase (2ʺ) gene has been developed as a new selectable marker for tobacco and many other monocots. Transformation has been accomplished effectively in soybean embryogenic cultures and tobacco suspension cell cultures but transformation of plastids in non-chloroplast tissues of cereal crops remains indefinable. To introduce foreign gene into plastid the most efficient technology till date is biolistic transformation. Due to the holistic approach of transplastomic technology, large amount of commercial transgenic plastids, like industrial and pharmaceutical proteins, metabolites, stress-tolerant plants, are likely to enter the marketplace and feed the world in the upcoming years.

Currently, selective plasmid DNA delivery via carbon nanotubes into the chloroplasts of some plants through the mechanism of lipid-exchange envelope-penetration without the aid of extraneous chemical or physical force has been designed. Though, further confirmation is needed to approve the use of this nanotechnology can be used for plastid transformation.

 References

Daniell, H., Lin, C. S., Yu, M., & Chang, W. J. (2016). Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome biology, 17(1), 1-29.

Li, S., Chang, L., & Zhang, J. (2021). Advancing organelle genome transformation and editing for crop improvement. Plant Communications, 100141.

Morley, S. A., & Nielsen, B. L. (2017). Plant mitochondrial DNA. Molecules, 15, 17.

 

Further reading

 

Johnston, I. G. (2019). Tension and resolution: dynamic, evolving populations of organelle genomes within plant cells. Molecular plant, 12(6), 764-783.

Johnston, I. G. (2019). Tension and resolution: dynamic, evolving populations of organelle genomes within plant cells. Molecular plant, 12(6), 764-783. 

Ruhlman, T. (2005). Chloroplast Biotechnology in Higher Plants: Expressing Antimicrobial Genes in the Plastid Genome.

Taunt, H. N., Stoffels, L., & Purton, S. (2018). Green biologics: The algal chloroplast as a platform for making biopharmaceuticals. Bioengineered, 9(1), 48-54. 

Tian, X., Zheng, J., Hu, S., & Yu, J. (2006). The rice mitochondrial genomes and their variations. Plant physiology, 140(2), 401-410.

 

 

 

 

 

 

 

 

 

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