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Review
. 2016 Jun 23;17(1):134.
doi: 10.1186/s13059-016-1004-2.

Chloroplast genomes: diversity, evolution, and applications in genetic engineering

Henry Daniell  1 , Choun-Sea Lin  2 , Ming Yu  3 , Wan-Jung Chang  2
Affiliations
Review

Chloroplast genomes: diversity, evolution, and applications in genetic engineering

Henry Daniell et al. Genome Biol. .

Abstract

Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.

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Figures

Fig. 1
Fig. 1
Map of the soybean (Glycine max) chloroplast genome. This genome was used to engineer biotic stress tolerance against insects and herbicides. The quadripartite structure includes two copies of an IR region (IRA and IRB) that separate large single-copy (LSC) and small single-copy (SSC) regions [18]. a Circular form. The GC content graph (gray circle inside) marks the 50 % threshold of GC content. b Linear form. Different colors indicate genes in different functional groups. IR inverted repeat, LSU large subunit, SSU small subunit
Fig. 2
Fig. 2
Chloroplast genome structure and gene expression across tracheophytes. These 658 chloroplast genomes were downloaded from NCBI Organelle Resources. The X-axis indicates the taxonomy of the chloroplast genome species following the Angiosperm Phylogeny Group III system and NCBI taxonomy. The bar width represents 100 species. The Y-axis shows the chloroplast genes, which were classified by different chloroplast regions. Gray boxes indicate absence of genes. Red boxes indicate stop codons in genes. Blue boxes indicate unknown nucleotides (N) in genes. IR inverted repeat, LSC large single-copy region, SSC small single-copy region
Fig. 3
Fig. 3
Basic process of chloroplast genetic engineering, diversity in intergenic spacer regions, and impact of transgene integration (endogenous versus heterologous genome sequences). a Complexity of heterologous sequence integration into intergenic spacer regions between lettuce and tobacco. The schematic diagram represents recombination between the tobacco transplastomic genome and the lettuce transformation vector [128]. Purple bars represent unique lettuce intron sequence; the green bar represents unique tobacco intron sequence; black bars are exon regions; blue regions are looped out sequence. The expression cassette comprises: promoters (P), leader sequence (L), gene of interest (GOI), terminators (T), and selectable marker gene (SMG). IG intergenic spacer region. b Basic process of chloroplast genetic engineering. Gene delivery is performed by bombardment with gold microparticles coated with chloroplast vectors, followed by three rounds of selection to achieve homoplasmy. After confirmation of transgene integration, plants are grown in the greenhouse to increase biomass. Chloroplast transgenes are maternally inherited without Mendelian segregation of introduced traits. c Comparison of 21 of the most variable intergenic spacer regions among Solanaceae chloroplast genomes. Atr Atropa, Pot potato, Tob tobacco, Tom tomato. *Tier 1, **tier 2, and ***tier 3 regions reported in the paper by Shaw et al. [250]. Plotted values were converted from percentage identity to sequence divergence on a scale from 0 to 1 as shown on the Y-axis; these values demonstrate a wide range of sequence divergence in different regions. Nucleotide sequences were determined by a bridging shotgun method and genome annotation was performed using the Dual Organellar GenoMe Annotator [13]. d, e Decrease in the expression of transgenes regulated by heterologous psbA promoters and untranslated regions (UTRs) engineered via tobacco chloroplast genomes. When the lettuce (La) psbA regulatory region was used in tobacco (Na) chloroplasts or vice versa, transgene expression is dramatically reduced. d Accumulation of a cholera toxin B subunit (CTB) and proinsulin (Pins) fusion protein (CP) was quantified by densitometry and e anthrax protective antigen (PA) accumulation was estimated by enzyme-linked immunosorbent assay (ELISA). Total leaf protein (TLP) or total soluble protein (TSP) data are presented as a function of light exposure and developmental stage. The order of young, mature, and old is different in d and e because of the accumulation of more CTB-Pins in older leaves and PA in mature leaves [128]. Young (top five), mature (fully grown), and old (bottom three) leaves were fully expanded and were cut from plants grown in the greenhouse for 8–10 weeks
Fig. 4
Fig. 4
Engineering the chloroplast genome to confer biotic/abiotic stress tolerance or expression of high-value products. a–d Industrial production of blood clotting factor IX (FIX) bioencapsulated in lettuce plants in a hydroponic cGMP facility. a Biomass production of FIX-expressing plants. b–d Steps in capsule preparation. After harvesting and lyophilization of fresh leaves, freeze-dried FIX-accumulating leaves were powdered and prepared as capsules [6]. e–g Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to the formation of the Bt insecticidal crystal protein. In bioassays with the Helicoverpa zea, f eating the transplastomic leaf kills the caterpillar, while g the control leaf is consumed by the growing caterpillar [137]. h Ultrastructure of the chloroplast envelope membrane of transplastomic γ-tocopherol methyltransferase (γ-TMT) tobacco plants shows the formation of multiple layers of inner envelope membranes as the result of γ-TMT overexpression [153]. i, j Expression of BETAINE ALDEHYDE DEHYDROGENASE (BADH) in carrot plants. i Transgenic carrot plants thrived in soil irrigated with 400 mM sodium chloride, whereas untransformed carrot plants showed retarded growth in the presence of salt. j Carrot roots from transplastomic plants [135]. k Phenotypes of tomato fruits from transplastomic tomato plants expressing lycopene β-cyclase transgenes compared with wild-type plants. Fruits were harvested at different ripening stages. Orange color of ripe fruits indicates efficient conversion of red lycopene into orange β-carotene (provitamin A) [154]
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