Open Access

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

Genome Biology201617:134

DOI: 10.1186/s13059-016-1004-2

Published: 23 June 2016

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.

Introduction

Chloroplasts are active metabolic centers that sustain life on earth by converting solar energy to carbohydrates through the process of photosynthesis and oxygen release. Although photosynthesis is often recognized as the key function of plastids, they also play vital roles in other aspects of plant physiology and development, including the synthesis of amino acids, nucleotides, fatty acids, phytohormones, vitamins and a plethora of metabolites, and the assimilation of sulfur and nitrogen. Metabolites that are synthesized in chloroplasts are important for plant interactions with their environment (responses to heat, drought, salt, light, and so on) and their defense against invading pathogens. So, chloroplasts serve as metabolic centers in cellular reactions to signals and respond via retrograde signaling [1, 2]. The chloroplast genome encodes many key proteins that are involved in photosynthesis and other metabolic processes.

The advent of high-throughput sequencing technologies has facilitated rapid progress in the field of chloroplast genetics and genomics. Since the first chloroplast genome, from tobacco (Nicotiana tabacum), was sequenced in 1986 [3], over 800 complete chloroplast genome sequences have been made available in the National Center for Biotechnology Information (NCBI) organelle genome database, including 300 from crop and tree genomes. Insights gained from complete chloroplast genome sequences have enhanced our understanding of plant biology and diversity; chloroplast genomes have made significant contributions to phylogenetic studies of several plant families and to resolving evolutionary relationships within phylogenetic clades. In addition, chloroplast genome sequences have revealed considerable variation within and between plant species in terms of both sequence and structural variation. This information has been especially valuable for our understanding of the climatic adaptation of economically important crops, facilitating the breeding of closely related species and the identification and conservation of valuable traits [4, 5]. Improved understanding of variation among chloroplast genomes has also allowed the identification of specific examples of chloroplast gene transfer to plant nuclear or mitochondrial genomes, which has shed new light on the relationship between these three genomes in plants.

In addition to improving our understanding of plant biology and evolution, chloroplast genomics research has important translational applications, such as conferring protection against biotic or abiotic stress and the development of vaccines and biopharmaceuticals in edible crop plants. Indeed, the first commercial-scale production of a human blood protein in a Current Good Manufacturing Processes (cGMP) facility was published recently [6]. The lack of conservation of intergenic spacer regions, even among chloroplast genomes of closely related plant species, and the species specificity of regulatory sequences have facilitated the development of highly efficient transformation vectors for the integration and expression of foreign genes in chloroplasts. Because the published literature is rarely cross-referenced, this review highlights the impact of chloroplast genomes on various biotechnology applications. In addition to our enhanced understanding of chloroplast biology, we discuss in depth the roles of chloroplast genome sequences in improving our understanding of intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes are engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In addition, we discuss the impact of chloroplast genome sequences on increasing our understanding of the origins of economically important cultivated species and changes that occurred during domestication.

Advances in chloroplast genome sequencing technology

One of the important factors in the rapid advancement of the chloroplast genomics field is improvement in sequencing technologies. In studies conducted before the availability of high-throughput methods, isolated chloroplasts were used for the amplification of the entire chloroplast genome by rolling circle amplification [712]. An alternative strategy is to screen bacterial artificial chromosome (BAC) or fosmid libraries using chloroplast genome sequences as probes [1320]; however, these methods are subject to many challenges, including difficulty in constructing good-quality BAC or fosmid libraries, large numbers of PCR reactions, and the possibility of contamination from other organellar DNA [2132]. The PCR approach is also difficult to apply to species that have no relatives whose chloroplast genomes have been sequenced or those with highly rearranged chloroplast genomes.

The development of next-generation sequencing (NGS) methods provided scientists with faster and cheaper methods to sequence chloroplast genomes. Moore and colleagues [33] first reported using NGS to determine chloroplast genome sequences, in Nandina and Platanus. Although multiple NGS platforms are available for chloroplast genome sequencing [34], Illumina is currently the major NGS platform used for chloroplast genomes [21, 32, 35, 36] because it allows the use of rolling circle amplification products [35, 37]. Investigators can then use bioinformatics platforms to perform de novo assembly without the need for reference genome sequences; from these assemblies it is possible to identify consensus chloroplast genome sequences [32]. A third-generation sequencer, the PacBio system which uses single molecule real-time (SMRT) sequencing, is now widely used in chloroplast genome sequencing [3843]. Its advantage is long read lengths [44], which facilitate de novo genome assembly, particularly in the four chloroplast junctions between the inverted repeat (IR) and single-copy regions.

The low accuracy (~85 % of the raw data) of the long reads produced by the PacBio platform [45] can be corrected by combining the latest chemistry with a hierarchical genome assembly process algorithm; accuracy rates as high as 99.999 % can be achieved after such post-error corrections [46]. Accuracy can also be increased using Illumina short reads [42]. In a study of Potentilla micrantha, sequencing with the Illumina platform produced seven contigs covering only 90.59 % of the chloroplast genome; by contrast, using the PacBio platform with error correction, the entire genome was successfully assembled in a single contig [39].

Chloroplast genome structure

The chloroplast genomes of land plants have highly conserved structures and organization of content; they comprise a single circular molecule with a quadripartite structure that includes two copies of an IR region that separate large and small single-copy (LSC and SSC) regions (Fig. 1a, b). The chloroplast genome includes 120–130 genes, primarily participating in photosynthesis, transcription, and translation. Recent studies have identified considerable diversity within non-coding intergenic spacer regions, which often include important regulatory sequences [13]. Despite the overall conservation in structure, chloroplast genome size varies between species, ranging from 107 kb (Cathaya argyrophylla) to 218 kb (Pelargonium), and is independent of nuclear genome size (Table 1). Certain lineages of land-plant chloroplast genomes also show significant structural rearrangements, with evidence of the loss of IR regions or entire gene families. Furthermore, there is also evidence for the existence of linear chloroplast genomes, as illustrated in Fig. 1b. The percentage of each form within the cell varies in different reports [47, 48].
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

Table 1

Alphabetical list of crop and tree species that have complete annotated chloroplast genome sequences

Species

Common name

Accession

Genome size (bp)

Uses

Reference(s)

Crops

     

Acorus gramineus

Sweet flag

NC_026299

152849

Ornamental

[169]

Agrostis stolonifera

Creeping bent grass

NC_008591

136584

Forage

[8]

Allium cepa

Onion

NC_024813

153538

Vegetable

[170]

Ananas comosus

Pineapple

NC_026220

159636

Fruit

[171]

Anthriscus cerefolium

Chervil

NC_015113

154719

Medicinal

[172]

Artemisia frigida

Fringed sagewort

NC_020607

151076

Medicinal

[173]

Atropa belladonna

Belladonna

NC_004561

156687

Medicinal

[174]

Brassica napus (2)*

Canola

NC_016734

152860

Oil

[175]

Calanthe triplicata

Black orchid

NC_024544

158759

Flower

[176]

Cannabis sativa (2)

Marijuana

NC_027223

153854

Fiber

[177]

Capsicum annuum (2)

Pepper

NC_018552

156781

Vegetable

[178]

Carica papaya

Papaya

NC_010323

160100

Fruit

[179]

Catharanthus roseus

Madagascar periwinkle

NC_021423

154950

Flower

[180]

Cenchrus americanus

Pearl millet

NC_024171

140718

Cereals

[181]

Cicer arietinum

Chickpea

NC_011163

125319

Vegetable

[7]

Coix lacryma-jobi

Job's tears

NC_013273

140745

Cereals

[29]

Colocasia esculenta

Taro

NC_016753

162424

Vegetable

[182]

Cucumis sativus (3)

Cucumber

NC_007144

155293

Vegetable

[183]

Curcuma roscoeana

Jewel of Burma

NC_022928

159512

Medicinal

[184]

Cymbidium tortisepalum (5)

Cymbidium orchid

NC_021431

155627

Flower

[55]

Cypripedium formosanum (3)

Formosa's lady's slipper

NC_026772

178131

Flower

[32]

Daucus carota

Carrot

NC_008325

155911

Vegetable

[9]

Dendrobium catenatum

Dendrobium orchid

NC_024019

152221

Flower

[56]

Dieffenbachia seguine

Dumbcane

NC_027272

163699

Ornamental

[185]

Digitaria exilis

White fonio

NC_024176

140908

Cereals

[181]

Echinochloa oryzicola

Late barnyard grass

NC_024643

139891

Cereals

[186]

Ephedra equisetina

Ma Huang

NC_011954

109518

Medicinal

[187]

Erycina pusilla

Mini orchid

NC_018114

143164

Flower

[36]

Fagopyrum esculentum (2)

Common buckwheat

NC_010776

159599

Cereals

[188]

Festuca arundinacea (4)

Kentucky fescue

NC_011713

136048

Forage

[189]

Fragaria vesca (6)

Wild strawberry

NC_015206

155691

Fruit

[190]

Glycine max (9)

Soybean

NC_007942

152218

Oil

[18]

Glycyrrhiza glabra

Common liquorice

NC_024038

127943

Medicinal

[74]

Gossypium barbadense (22)

Sea island cotton

NC_008641

160317

Fiber

[69]

Guizotia abyssinica

Ramtilla

NC_010601

151762

Bird seed

[191]

Helianthus annuus (9)

Common sunflower

NC_007977

151104

Oil

[192]

Heliconia collinsiana

Platanillo

NC_020362

161907

Ornamental

[193]

Hordeum vulgare

Barley

NC_008590

136462

Cereals

[8]

Hyoscyamus niger

Henbane

NC_024261

155720

Medicinal

[194]

Ipomoea batatas

Sweet potato

NC_026703

161303

Vegetable

[195]

Ipomoea purpurea

Common morning glory

NC_009808

162046

Ornamental

[196]

Lactuca sativa

Lettuce

NC_007578

152765

Vegetable

[197]

Lilium superbum

Turk's-cap lily

NC_026787

152069

Flower

[198]

Lolium multiflorum (2)

Ryegrass

NC_019651

135175

Lawn

[199]

Lotus japonicus

Birdsfoot trefoil

NC_002694

150519

Forage

[200]

Manihot esculenta

Cassava

EU117376

161453

Starch crop

[20]

Masdevallia picturata (2)

Masdevallia orchid

NC_026777

157423

Flower

[32]

Musa textilis

Banana

NC_022926

161347

Fruit

[184]

Nicotiana tabacum (4)

Tobacco

Z00044

155943

Tobacco

[3]

Nuphar advena

Spatterdock

NC_008788

160866

Medicinal

[201]

Nymphaea alba (2)

White water-lily

NC_006050

159930

Flower

[24]

Oncidium hybrid

Oncidium

NC_014056

146484

Flower

[54]

Oryza sativa (6)

Rice

X15901

134525

Cereals

[202]

Panax ginseng (2)

Ginseng

NC_006290

156318

Medicinal

[203]

Panicum virgatum

Switchgrass

NC_015990

139619

Biofuel

[204]

Paphiopedilum armeniacum (2)

Slipper orchid

NC_026779

162682

Flower

[32]

Parthenium argentatum

Guayule

NC_013553

152803

Biofuel

[205]

Pelargonium (2)

Geranium

NC_008454

217942

Flower

[206]

Phalaenopsis hybrid (3)

Phalaenopsis orchid

NC_007499

148964

Flower

[51]

Phaseolus vulgaris

Kidney bean

NC_009259

150285

Bean

[78]

Pisum sativum

Pea

NC_014057

122169

Vegetable

[76]

Raphanus sativus

Radish

NC_024469

153368

Vegetable

[207]

Ravenala madagascariensis

Traveller's tree

NC_022927

166170

Ornamental

[184]

Ricinus communis

Castor bean

NC_016736

163161

Oil

[208]

Saccharum hybrid (2)

Sugarcane

NC_005878

141182

Sugar

[209]

Salvia miltiorrhiza

Redroot sage

NC_020431

151328

Medicinal

[210]

Secale cereale

Rye

NC_021761

114843

Cereals

[64]

Sesamum indicum

Sesame

NC_016433

153324

Oil

[211]

Solanum lycopersicum (11)

Tomato

NC_007898

155461

Vegetable

[13]

Solanum tuberosum

Potato

DQ231562

155312

Starch crop

[212]

Sorghum bicolor (2)

Sorghum

NC_008602

140754

Cereals

[8]

Spinacia oleracea

Spinach

NC_002202

150725

Vegetable

[213]

Trifolium grandiflorum (8)

Large-flower hop clover

NC_024034

125628

Forage

[74]

Triticum aestivum (6)

Bread wheat

NC_002762

134545

Cereals

[63]

Vanilla planifolia

Vanilla

NC_026778

148011

Fruit

[32]

Vigna radiata (3)

Mung bean

NC_013843

151271

Bean

[79]

Zea mays

Maize

NC_001666

140384

Cereals

[62]

Zingiber spectabile

True ginger

NC_020363

155890

Ornamental

[193]

Trees and perennial plants

     

Abies koreana

Fir

NC_026892

121373

Wood

[214]

Actinidia chinensis (2)

Kiwifriut

NC_026690

156346

Fruit

[215]

Amentotaxus formosana

Taiwan catkin yew

NC_024945

136430

Timber

[216]

Araucaria heterophylla

Norfolk island araucaria

NC_026450

146723

Timber

[217]

Bambusa multiplex (4)

Golden goddess bamboo

NC_024668

139394

Ornamental

[91]

Bambusa oldhamii

Green bamboo

NC_012927

139350

Vegetable

[28]

Berberis bealei

Beale's mahonia

NC_022457

164792

Ornamental

[218]

Bismarckia nobilis

Bismarck palm

NC_020366

158210

Ornamental

[193]

Buxus microphylla

Japanese box

NC_009599

159010

Ornamental

[219]

Calocedrus formosana

Taiwan incense-cedar

NC_023121

127311

Timber

[220]

Calycanthus floridus

Carolina-allspice

NC_004993

153337

Medicinal

[23]

Camellia oleifera (13)

Tea oil plant

NC_023084

156971

Oil

[221]

Camellia reticulata

To-tsubaki

NC_024663

156971

Flower

[222]

Carludovica palmata

Toquilla palm

NC_026786

158545

Fiber

[198]

Castanea mollissima

Chestnut

NC_014674

160799

Fruit

[14]

Cathaya argyrophylla

Cathaya

NC_014589

107122

Timber

[223]

Cedrus deodara

Cedar

NC_014575

119299

Timber

[223]

Cephalotaxus wilsoniana (2)

Wilson plum yew

NC_016063

136196

Timber

[224]

Chrysobalanus icaco

Coco plum

NC_024061

162775

Fruit

[225]

Citrus sinensis (2)

Orange

NC_008334

160129

Fruit

[12]

Cocos nucifera

Coconut

NC_022417

154731

Oil

[226]

Coffea arabica

Coffee

NC_008535

155189

Beverage

[10]

Corymbia gummifera (4)

Red bloodwood

NC_022407

160713

Timber

[227]

Corynocarpus laevigata

Karaka nut

NC_014807

159202

Fruit

[37]

Cryptomeria japonica

Sugi

NC_010548

131810

Timber

[228]

Dendrocalamus latiflorus

Sweet giant bamboo

NC_013088

139394

Vegetable

[28]

Elaeis guineensis

African oil palm

NC_017602

156973

Oil

[229]

Eucalyptus globulus (32)

Eucalyptus

NC_008115

160286

Timber

[230]

Hevea brasiliensis

Rubber tree

NC_015308

161191

Rubber

[231]

Jasminum nudiflorum

Winter jasmine

NC_008407

165121

Ornamental

[232]

Jatropha curcas

Barbados nut

NC_012224

163856

Biofuel

[233]

Juniperus bermudiana (4)

Bermuda juniper

NC_024021

127659

Timber

[234]

Larix decidua

European larch

NC_016058

122474

Timber

[224]

Licania sprucei (3)

Licania

NC_024065

162228

Ornamental

[225]

Liquidambar formosana

Chinese sweetgum

NC_023092

160410

Timber

[30]

Liriodendron tulipifera

Tulip tree

NC_008326

159886

Timber

[235]

Metasequoia glyptostroboides

Dawn redwood

NC_027423

131887

Timber

[236]

Millettia pinnata

Indian beech

NC_016708

152968

Ornamental

[81]

Morus indica (3)

White mulberry

NC_008359

158484

White mulberry

[237]

Nageia nagi

Asian bayberry

NC_023120

133722

Timber

[220]

Nandina domestica

Heavenly bamboo

NC_008336

156599

Ornamental

[33]

Nerium oleander

Oleander

NC_025656

154903

Ornamental

[238]

Olea europaea (5)

Olive

NC_015604

155862

Oil

[239]

Phoenix dactylifera

Date palm

NC_013991

158462

Fruit

[240]

Phyllostachys edulis (4)

Moso bamboo

NC_015817

139679

Timber

[89]

Picea sitchensis (3)

Sitka spruce

NC_011152

120176

Timber

[35]

Pinus taiwanensis (12)

Taiwan red pine

NC_027415

119741

Timber

[241]

Platanus occidentalis

American sycamore

NC_008335

161791

Ornamental

[33]

Podocarpus lambertii (3)

Podocarpus

NC_023805

133734

Ornamental

[242]

Populus alba

White poplar

NC_008235

156505

Timber

[243]

Prinsepia utilis

Himalayan cherry

NC_021455

156328

Ornamental

[244]

Prunus persica (6)

Peach

NC_014697

157790

Fruit

[14]

Pseudophoenix vinifera

Florida cherry palm

NC_020364

157829

Ornamental

[193]

Pseudotsuga sinensis

Chinese douglas

NC_016064

122513

Timber

[224]

Pyrus pyrifolia (2)

Chinese pear

NC_015996

159922

Fruit

[245]

Quercus rubra (4)

Oak

NC_020152

161304

Timber

[246]

Sapindus mukorossi

Soapberries

NC_025554

160481

Medicinal

[247]

Taiwania cryptomerioides (2)

Taiwania

NC_016065

132588

Timber

[224]

Theobroma cacao

Cacao tree

HQ336404

160604

Beverage

[14]

Vaccinium macrocarpon

Large cranberry

NC_019616

176045

Fruit

[248]

Vitis vinifera

Wine grape

NC_007957

160928

Fruit

[19]

Wollemia nobilis

Wollemia

NC_027235

145630

Timber

[249]

*The number of species in the same genus as the listed species that have sequenced and annotated chloroplast genomes is shown in parentheses

Like the genes, the introns in land-plant chloroplast genomes are generally conserved, but the loss of introns within protein-coding genes has been reported in several plant species [49], including barley (Hordeum vulgare) [8], bamboo (Bambusa sp.) [28], cassava (Manihot esculenta) [20], and chickpea (Cicer arietinum) [7]. The proteins encoded by genes in which intron loss is known to occur have diverse functions; they include an ATP synthase (atpF), a Clp protease (clpP), an RNA polymerase (rpoC2), and ribosomal proteins (rpl2, rps12, and rps16) [49]. The majority of reported intron losses have been observed in specific plant groups or species, although some examples of intron loss (such as that in clpP) occur in diverse plant species, including monocots (Poaceae), eudicots (Onagraceae and Oleaceae) and gymnosperms (Pinus) [49].

Diversity of chloroplast genome sequences

At higher taxonomic levels (family level), protein-coding regions and conserved sequences of the chloroplast genome can be used for phylogenetic analysis and domestication studies [49]. Earlier phylogenetic analyses utilized partial chloroplast DNA sequences. The use of variable regions or multiple DNA fragments dramatically enhanced the utility of these analyses but there is insufficient information in these sequences to provide the high-resolution necessary to differentiate closely related taxa, particularly some within-species taxa whose taxonomic relationships are unclear. Complete chloroplast genome sequences are valuable for deciphering phylogenetic relationships between closely related taxa and for improving our understanding of the evolution of plant species.

In this section, we discuss several examples of comparisons of chloroplast genomes, within and between crop species, that have provided unique insight into evolutionary relationships among taxa. We also discuss the origin and geographic distribution of economically important species, as well as their adaptations to different climatic conditions and the use of genome information in their breeding and conservation.

A key application of the chloroplast genome in agriculture is the identification of commercial cultivars and the determination of their purity. DNA barcodes derived from the chloroplast genome can be used to identify varieties and in the conservation of breeding resources. Success in breeding is determined by genetic compatibility and chloroplast genomes serve as a valuable tool for identifying plants that are likely to be closely related and, therefore, genetically compatible. Understanding the genetic relationships between cultivated crops and their wild relatives informs efforts to introduce specific advantageous traits into cultivated crops. In the section below, we discuss how chloroplast genomes have been used to elucidate the evolutionary relationships and domestication history of a few major crops and how this informs breeding programs.

Breeding

The Orchidaceae is a large family that encompasses about 6–11 % of all angiosperms [50] and is important in floriculture. Many commercially important orchid species belong to the subfamily Epidendroideae and chloroplast genomes of several species from this subfamily have been sequenced [5158]. Because it is easy to perform inter-generic crossing in orchids and because the record of breeding is sometimes incomplete, it is often difficult to validate the parental origin of commercially important varieties [54]. Corrected parental information is important for breeding and variety identification. In an investigation of the Oncidiinae, a subtribe within the Epidendroideae, PCR products derived from eight conserved regions in 15 commercial varieties resolved their phylogenetic relationship at the species level [54] and helped to resolve putative errors in parental origin. Parental records had indicated that Odontoglossum ‘Violetta von Holm’, Odontoglossum ‘Margarete Holm’ and Odontocidium ‘Golden Gate’ are derived from the same female parent (Odontoglossum bictoniense) but phylogenetic analyses of ‘Violetta von Holm’ did not correlate with those of ‘Golden Gate’ or ‘Margarete Holm’ [54]. A possible reason for inconsistencies between the chloroplast DNA-based phylogenetic tree and the parental record is chloroplast capture. Chloroplast capture is the introgression of chloroplasts from one species into another after intrageneric and intergeneric hybridization [59]. Although chloroplast genomes provide useful information for phylogenetic analyses involving closely related taxa, chloroplast capture by hybridization may distort phylogenetic relationships if captured chloroplast genomes or genes included therein are used [60]. The use of both nuclear and chloroplast genomes can provide more complete phylogenies [4, 61].

Phylogenetic studies

There are several published chloroplast genomes from cereals, including those from sorghum (Sorghum bicolor), barley [8], maize (Zea mays) [62], wheat (Triticum aestivum) [63], rye (Secale cereale) [64], and rice (Oryza sativa) [65]. Rice is one of the world's most important crops and is the primary carbohydrate source for the global human population (http://www.ers.usda.gov/topics/crops/rice.aspx). The Oryza species are classified into ten genome types, including six diploids (AA, BB, CC, EE, FF, and GG) and four allotetraploids (BBCC, CCDD, HHJJ, and HHKK). Attempts to clarify the evolutionary relationships between cultivated rice and its wild relatives remain contentious and inconclusive [4]. For example, there are two wild species that have an AA genome in Australia, Oryza meridionalis (annual) and Oryza rufipogon (perennial). Oryza sativa was domesticated from Asian O. rufipogon 10,000 years ago [65]. Nevertheless, analysis of complete Australian and Asian wild rice chloroplast genomes indicated that Australian O. rufipogon chloroplast genomes are more similar to those of Australian O. meridionalis than to those of Asian O. rufipogon [6567]. Using 19 chloroplast genomes of Oryza AA genome species, a robust phylogenetic tree was established, which will aid in improving rice crops and in conservation strategies [4, 5].

Cotton is the most important textile fiber crop and the first cotton (Gossypium hirsutum) chloroplast genome was published in 2006 [11]. The diploid Gossypium species comprise eight genome groups (A to G and K genomes). Gossypium hirsutum (upland cotton), the most widely planted cotton species in the world, is an allotetraploid of the ancestral A and D genome species [68]. Chloroplast genome sequences are available for 22 Gossypium species and these can be used to glean information about the evolution and domestication of this crop [11, 68, 69] (Table 1). Simple sequence repeat primers were used to investigate 41 species of Gossypium, including all eight genome groups and allotetraploid species [70]. The results indicated that two modern A-genome species, Gossypium herbaceum and Gossypium arboretum, were not cytoplasmic donors of tetraploid (AD) species; instead, the AD genome species originated from an extinct ancestor species of the modern A genome [68, 70].

Domestication

Information on chloroplast genomes is useful for understanding the domestication of several crops, particularly legumes [71]. The chloroplast genome structure of legumes is very interesting; it contains multiple rearrangements, including large inverted segments and loss of inverted repeats [72]. An example is a 51-kb inversion that was first identified in the soybean (Glycine max) chloroplast genome sequence [18] before being reported in most members of the subfamily Papilionoideae [7, 7377]. A 78-kb reversion was subsequently confirmed in Phaseolus and Vigna chloroplast genomes [78, 79]. More recently, 36-kb [80] and 5.6-kb [81] inversions inside the 51-kb inversion were identified. There are many important genes within these inverted regions but no gene is disturbed and plant survival and performance are not affected. These unique characteristics are not only very useful in phylogenetic studies [82] but also provide important information for chloroplast transformation in legumes. Chloroplast structure is also important for the design of primers needed in the amplification of sequences for further domestication and phylogenetic analysis.

Citrus is one of the most commercially important fruit genera. In 2006, the first Citrus chloroplast genome, that of sweet orange (Citrus × sinensis), was published [12] and this served as a reference genome for subsequent publications [83, 84]. Phylogenetic analysis of 34 chloroplast genomes of Citrus (28) and Citrus-related genera (6) indicated that citrus fruits have the same common ancestor [84, 85]. In four genes (matK, ndhF, ycf1, and ccsA), single-nucleotide variations and insertion/deletion frequencies were clearly higher than average and showed that these genes have been positively selected. The matK gene encodes a maturase that is involved in splicing type II introns and the matK sequence is often used in phylogenetic and evolutionary studies [84]. Positive selection of matK is observed not only in citrus but is common in several other plant species. In fact, more than 30 plant groups have been shown to undergo positive selection of matK genes, indicating that the gene is subject to a number of different ecological selective pressures [86]. The ndhF gene encodes a subunit of the chloroplast NAD(P)H dehydrogenase (NDH) complex. Chloroplast NDH monomers are sensitive to high light stress, suggesting that the ndh genes may also be involved in stress acclimation [87]. These studies indicated that matK and ndhF show positive selection in Australian species, potentially contributing to their adaptation to a hot, dry climate [84, 85].

Bamboo is an economically and ecologically important forest plant in Asia [88]. Bamboo grows quickly and new culms are regenerated from the rhizome after harvesting, making it a sustainable and ecologically and environmentally friendly crop. The first two bamboo chloroplast genomes have been published [28] and many more bamboo chloroplast genomes are now available [8893]. Bamboo has a long juvenility and it is difficult to obtain flowers for taxonomic studies; consequently the taxonomic relationships of bamboo have proven challenging to unravel on the basis of traditional reproductive organ morphology. Furthermore, the extremely low rate of sequence divergence meant that the taxonomic and phylogenetic relationships of temperate woody bamboos at lower taxonomic levels proved difficult to resolve [88]. These relationships were eventually resolved with high-resolution phylogenetic trees using 25 bamboo chloroplast genomes [93]. In addition to woody bamboos, chloroplast genomes have also been published for herbaceous bamboo [88, 92]. An interesting phenomenon identified in herbaceous bamboo chloroplast genomes is that of gene transfer from the mitochondrial genome to the chloroplast genome. This was an unusual observation, as the chloroplast genome is thought to be nearly immune to the transfer of DNA from nuclear and mitochondrial genomes [88, 92, 94]. A possible reason for this recalcitrance to DNA transfer is the lack of an efficient DNA uptake system [94]. Prior to its observation in herbaceous bamboo, this phenomenon was only observed in two eudicot chloroplast genomes [94] and in monocots [88, 92].

Transfer of chloroplast genes to nuclear or mitochondrial genomes

There are three distinct genomes in plant cells: nuclear, mitochondrial, and plastid. Mitochondria are believed to have evolved from a single endosymbiotic event by the uptake of a proteobacterium, whereas chloroplasts evolved from endosymbiosis of a cyanobacterium, after which there was a massive transfer of genes from the chloroplast to the nucleus [95]. There are distinct translation systems in these organelles: nuclear-encoded genes are translated in the cytosol and the protein products are then transported to the locations in which they function, including chloroplasts [96], whereas chloroplast-encoded proteins are directly synthesized within the chloroplast. Multi-subunit functional protein complexes that are involved in photosynthesis or protein synthesis are also assembled within chloroplasts.

Gene content, number, and structure are conserved in the chloroplast genome sequences of most autotrophic land plants [97, 98] but some protein-encoding genes are absent in specific species [49]. The loss of genes such as infA, rpl22, and ndh from the chloroplast genome and their intracellular transfer to the nuclear or mitochondrial genomes provide valuable information for phylogenetic analyses and evolutionary studies. It is very easy to identify the chloroplast origin of genes in plant mitochondrial or nuclear genomes [99, 100] by intracellular gene transfer [32], but this could also lead to erroneous phylogenic relationships when short sequences are used instead of complete chloroplast genome sequences.

The chloroplast translation initiation factor 1 (infA) is a homolog of the essential gene infA in Escherichia coli [101, 102]. This gene initiates translation in collaboration with two nuclear-encoded initiation factors to mediate interactions between mRNA, ribosomes, and initiator tRNA-Met [102]. Many parallel losses of chloroplast-encoded infA have occurred during angiosperm evolution [102] (Fig. 2). Nuclear-encoded infA genes have been identified in Arabidopsis thaliana, soybean, tomato (Solanum lycopersicum), and ice plant (Mesembryanthemum crystallinum) [102]. Protein sequences of nuclear-encoded infA in these four species contain chloroplast transit peptides. Studies using soybean and A. thaliana infA-GFP proteins have shown that nuclear-encoded infA genes are translated in the cytosol and transported into chloroplasts [102]. Many more chloroplast-encoded infA deletions have been identified recently (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

There are 57 chloroplast genomes in 26 genera in which the essential gene rpl22 is reported to have been deleted from the chloroplast and transferred to the nuclear genome (Fig. 2) [14, 103]. Nuclear-encoded rpl22 contains a transit peptide that is predicted to deliver this protein from the cytosol to chloroplasts. These peptides are diverse, suggesting that there were two independent rpl22 transfers in the Fabaceae and the Fagaceae [14]. Similar transfer to the nucleus has also been observed for rpl32 deletion from chloroplast genomes [104106].

Eleven chloroplast genes encode ndh subunits, which are involved in photosynthesis. The ndh proteins assemble into the photosystem I complex to mediate cyclic electron transport in chloroplasts [107, 108] and facilitate chlororespiration [109]. Some autotrophic plants lack functional ndh genes in their chloroplast genomes [36, 51, 54, 55, 110115] (Fig. 2). Unlike the single gene losses described previously, the entire family of ndh genes has been deleted in these plants. Seven orchid chloroplast genomes indicated at least three independent ndh deletions [32]. Some orchid ndh DNA fragments were identified in the mitochondrial genome but the complete ndh genes required to translate putative functional protein complexes are absent [32]. In the nuclear genome of Norway spruce, only non-functional plastid ndh gene fragments are present [116]. Normal photosynthesis is observed in these ndh-deleted species [32, 117]. Furthermore, ndh-deleted transformants are autotrophic and produce carbohydrates through photosynthesis [107, 118121].

Many more chloroplast-gene deletions have been observed, including deletions of accD, ycf1, ycf2, ycf4, psaI, rpoA, rpl20, rpl23, rpl33, and rps16; many unique gene deletions have been identified in only one or a few species (psbJ, rps2, rps14, and rps19) (Fig. 2). The functions of these genes, phenotypes of their knock-out mutants, and evidence for their transfer are summarized in Additional file 1. Most essential genes that have been lost from chloroplast genomes have been transferred to the nucleus to maintain the plant's photosynthetic capacity, with the exception of ycf1 and ycf2.

In summary, chloroplast genome sequences are most valuable for understanding plant evolution and phylogeny. Databases of not only plant genomes but also plant transcriptomes will be useful in investigating deletion events or the transfer of chloroplast genes to other organellar genomes to complement such deletions.

Advances in chloroplast genome engineering

In the past century, desirable agronomic traits, including yield enhancement and resistance to pathogens or abiotic stress, were achieved by breeding cultivated crops with their wild relatives. As explained above, chloroplast genome sequences are very useful in the identification of closely related, breeding-compatible plant species. With the advent of modern biotechnology, desirable traits from unrelated species can now be readily introduced into commercial cultivars. Such genetically modified crops have revolutionized agriculture in the past two decades, dramatically reducing the use of chemical pesticides and herbicides while enhancing yield. For most commercial cultivars, herbicide- or insect-resistance genes are introduced into the nuclear genome. There are, however, a few limitations for nuclear transgenic plants, including low levels of expression (<1 % total soluble protein (TSP)) and potential escape of transgenes via pollen.

Engineering the introduction of foreign genes into the chloroplast genome addresses both of these concerns. Just two copies of transgenes are typically introduced into the nuclear genome, whereas up to 10,000 transgene copies have been engineered into the chloroplast genome of each plant cell, resulting in extremely high levels of foreign gene expression (>70 % TSP) [122]. Most importantly, chloroplast genomes are maternally inherited in most cultivated crops, minimizing or eliminating transgene escape via pollen [123].

The basic process of chloroplast engineering is explained in Fig. 3a, b. Chloroplast genome engineering is accomplished by integrating foreign genes into intergenic spacer regions without disrupting the native chloroplast genes (Fig. 3a). Two chloroplast genes are used as flanking sequences to facilitate integration of transgene cassettes. Transgene cassettes include a selectable marker gene and gene(s) of interest, both regulated by chloroplast gene promoters and untranslated regions (UTRs; Fig. 3a). Chloroplast genome sequences are essential to build transgene cassettes because they provide both flanking and regulatory sequences. Transgene cassettes that are inserted into bacterial plasmids are called chloroplast vectors and they are bombarded into plant cells using gold particles and a gene gun (Fig. 3b). Because of the presence of chloroplast DNA in the nuclear or mitochondrial genome, transgene cassettes may integrate via homologous or non-homologous recombination events; but any transgenes that are integrated within the nuclear or mitochondrial genome will not be expressed because chloroplast regulatory sequences are not functional in other genomes. If such integration occurs, the transgenes could be easily identified by evaluation of their integration site and eliminated [124].
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

One of the challenges of creating chloroplast transgenic (transplastomic) plants is the elimination of all untransformed copies (>10,000 per cell) of the native chloroplast genome and replacing them with transformed genomes that contain integrated transgene cassettes. The absence of the native chloroplast genome and the presence of only the modified genomes is referred to as the homoplasmic state, which is typically achieved after two or three rounds of selection (Fig. 3b). The most effective selectable marker used is the aadA gene, which confers resistance to streptomycin and spectinomycin. These antibiotics bind specifically to chloroplast ribosomes and disrupt protein synthesis without interfering with any other cellular process. Efforts to transform the chloroplast genome of cereal crops have been mostly unsuccessful. This could be due to the instability of chloroplast DNA in the mature leaves of cereals [47] or to a requirement for better selectable markers [125].

Table 2 provides the first global, comprehensive summary of the power of chloroplast genetic engineering, utilizing valuable information generated by the sequencing of chloroplast genomes described in previous sections. This table includes the most complete list of chloroplast genomes that have been engineered for enhanced agronomic traits or the production of different bio-products, including biopolymers, industrial enzymes, biopharmaceuticals, and vaccines. Within Table 2, transgenes are grouped according to their functions and are organized according to their site of integration. The efficiency of transgene expression is also included in Table 2, providing important information about the regulatory sequences used to express the transgenes.
Table 2

Engineering the chloroplast genome for biotechnology applications

Site of integration

Transgenes

Regulatory sequences

Efficiency of expression

Engineered traits or products

Reference(s)

Insect or pathogen tolerance

trnI/trnA

Bgl-1

5′psbA/3′psbA

>160-fold enzyme

Resistance against whitefly and aphid

[141]

trnI/trnA

Pta

5′psbA/3′psbA

7.1–9.2 % TSP

Broad-spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens

[142]

trnI/trnA

PelB1, PelD2

5′psbA/3′psbA

~2.42 units mg−1 FW

Resistance against Erwinia soft rot

[150]

trnI/trnA

RC1011, PG12

5′psbA/3′

17–38 % TSP

Resistance to Erwinia soft rot and tobacco mosaic virus

[140]

trnI/trnA

cpo

Prrn/psbA/psbA

NR

Resistance to fungal pathogens in vitro (Aspergillus flavus, Fusarium verticillioides, and Verticillium dahliae) and in planta (Alternaria alternata)

[251]

trnI/trnA

Bt cry2Aa2 operon

Prrn/ggagg/psbA

45.3 % TSP

100 % mortality of cotton bollworm, beet armyworm; cuboidal Bt crystals formation

[137]

trnI/trnA

Bt cry9Aa2

Prrn/ggagg/rbcL

~10 % of TSP

Resistance to Phthorimaea operculella

[252]

trnI/trnA

msi-99

Prrn/ggagg/psbA

21–43 % TSP

Resistance to in planta challenge of Pseudomonas syringae, Aspergillus flavus, Fusarium moniliforme, Verticillium dahlia, and Colletotrichum destructivum

[253]

trnI/trnA

sporamin1, CeCPI2, and chitinase2

Prrn/TpsbA

0.85–1 % TSP

Resistance against Spodoptera litura and Spodoptera exigua leaf spot, as well as soft rot diseases

[254]

trnI/trnA

MSI-99

Prrn/Trps16

89.75 μg g−1 FW

Resistance against rice blast fungus

[255]

trnV/rps12/7

cry1A(c)

Prrn/rbcL/rps16

3–5 % of TSP

Resistance to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua

[256]

trnV/rps12/7

cry1Ab

Prrn/T7gene10/rbcL

NR

Resistance to caterpillar of Anticarsia gemmatalis

[145]

rbcL/accD

cry2Aa2

Prrn/ggagg/psbA

2–3 % of TSP

Resistance to Heliothis virescens, Helicoverpa zea, and Spodoptera exigua

[257]

Abiotic stress tolerance

trnI/trnA

tps1

Prrn/ggagg/psbA

>169-fold transcript

Drought tolerance: growth in 6 % polyethylene glycol and rehydration after 24 days of drought

[258]

trnI/trnA

merA/merB

Prrn/ggagg/psbA

NR

Phytoremediation: high level tolerance to the organomercurial compounds, up to 400 μM phenylmercuric acetate

[259]

trnI/trnA

badh

Prrn/T7 g10/rps16

93–101 μM g−1 FW

Salt tolerance: carrot plants survived up to 400 mM NaCl

[135]

trnI/trnA

γ-TMT

Prrn/T7g 10/TpsbA

>7.7 % TSP

Increased salt and heavy metal tolerance, enhanced accumulation of ɑ-tocopherol in seeds

[153]

trnI/trnA

mt1

Prrn/T7 g10/Trps16

NR

Phytoremediation: resistant to mercury, up to 20 μm

[260]

trnV/rps12/7

b-bar1

Prrn/TrbcL

>7 % TSP

Resistance to the herbicide phosphinothricin

[261]

trnV/rps7/12

EPSPS

Prrn/Trps16

>10 % TSP

Resistance to the herbicide glyphosate

[262]

rbcL/accD

EPSPS/aroA

Prrn/ggagg/psbA

NR

Resistance to glyphosate (>5 mM)

[129]

rbcL/accD

mALS

PpsbA/TpsbA

NR

Tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides

[263]

rbcL/accD

Bar

Prrn/rbcL/psbA

NR

Herbicide resistance: up to 25 μg ml−1 glufosinate

[264]

rbcL/rbcL

Hppd

psbA/psbA/3′rbcL

5 % TSP

Resistance to herbicide

[265]

rbcL/accD

panD

Prrn/rbcL 3′

>4-fold β-alanine

Tolerance to high-temperature stress

[266]

trnfM/trnG

lycopene β-cyclase

atpI/rps16

0.28 mg g−1 DW

Herbicide resistance and triggers conversion of lycopene

[133]

prs14/trnG

HTP, TCY, TMT

Prrn/T7 g10/TrbcL

NR

Increase in vitamin E in fruit; cold-stress tolerance

[267]

Other agronomic traits

trnI/trnA

phaA

Prrn/psbA/psbA

14.71β-ketothiolase mg−1 FW

Engineered cytoplasmic male sterility

[268]

trnI/trnA

RbcS

T7g10 or psbA

>150-fold RbcS transcript

Restoration of RuBisCO activity in rbcS mutants

[136]

rbcL/accD

TC, γ -TMT

PpsbA/Trsp16

3 nmol h−1 mg−1 FW

Vitamin E accumulation in tobacco and lettuce

[269]

rbcL/accD

CrtZ, CrtW

Prrn/Trps16

NR

Accumulation of astaxanthin fatty acid esters in lettuce

[270]

trnV/orf708

BicA

psbA/psbA/psbA

~0.1 % TSP

CO2 capture within leaf chloroplasts

[271]

trnV/3′rps12

Trx f, Trx m

prrn T7G10/rps12

NR

Starch synthesis/chloroplast redox regulation

[272]

trnfM/trnG

CV-N

Prrn/T7g10/TatpA

~0.3 % TSP

Increased mRNA stability and protein stability with the expression of CV-N in chloroplasts

[273]

trnI/trnA

Bgl-1

5′psbA/3′psbA

44.4 units Bgl1 g−1 FW

β-Glucosidase increased enzyme cocktail efficiently to release sugar from paper, citrus peel, and wood

[141]

trnI/trnA

ubiC

5′psbA/3′psbA

25 % DW

250-fold higher pHBA polymer accumulation than nuclear transgenic lines

[149]

trnI/trnA

man 1

5′psbA/3′psbA

25 units g−1 FW

Mannanase increased enzyme cocktail released sugar from paper, citrus peel, and wood

[274]

trnI/trnA

cutinase or swoIlenin

5′PsbA/3′PsbA

47.7 % reduction of MGDG and DGDG in cutinase and 68.5 % in swollenin

Swollenin enlarged and irreversibly unwound cotton fiber; cutinase showed esterase and lipase activity; used in enzyme cocktails

[275]

trnI/trnA

bgl1

5′psbA/3′psbA

14 units mg−1 FW

Enzyme cocktails produced glucose from filter paper, pine wood, or citrus peel

[150]

swo1

NR

xyn2

421 units mg−1 FW

Acetyl sylan esterase

NR

celD

493 units mg−1 FW

celO

442 units mg−1 FW

Lipase

NR

Cutinase

15 units mg−1 FW

trnI/trnA

PMK, MVK, MDD, AACT, HMGS, HMGRt; IPP, FPP, ADS, CYP71AV1, AACPR

Prrn/PpsbA

0.1 mg g−1 FW

Artemisinic acid for several isoprenoid products

[276]

trnI/trnA

Cel6A,Cel6B

Prrn/rbcL/rbcL

2–4 % TSP

Hydrolyzed crystalline cellulose

[277]

trnfM/trnG

bgl1C, cel6B, cel9A, xeg74

Prrn/T7g10/TrbcL

5– 40 % TSP

Cell wall-degrading enzyme activity

[278]

rbcL/accD

phbC, phbA, phbB

Prrn/rbcL 3′

0.16 % DW

Polyhydroxybutyrate (PHB) accumulation in leaves

[279]

rbcL/accD

crtZ, crtW

Prrn/Trps16

>0.5 % DW

Astaxanthin accumulation

[280]

trnV/rps7

EGPh

psbA/psbA/Trps16

25 % TSP

Chloroplast-derived β-1,4-endoglucanase (EGPh) was recovered from dry leaves and digested carboxymethyl cellulose (CMC) substrate

[281]

trnI/trn A

EX4

PpsbA/TpsbA

14.3 % TSP

CTB–EX4 showed increased insulin secretion similar to the commercial injectable EX4 in pancreatic β-cells and in mice fed with cells expressing EX4 in chloroplasts

[160]

trnI/trn A

MBP

PpsbA/TpsbA

2 % TSP

Amyloid loads were reduced in ex vivo studies in human Alzheimer’s brain and in vivo in Alzheimer’s mice fed with bio-encapsulated CTB–MBP. Abeta was also reduced in retinae and loss of retinal ganglion cells was prevented

[162]

trnI/trn A

FVIII

PpsbA/TpsbA

370 mg g−1 FW

Feeding of the HC/C2 antigen mixture substantially suppressed T-helper cell responses and inhibitor formation against FVIII in hemophilia A mice

[282]

trnI/trn A

HSA

PpsbA/TpsbA

26 % TSP

In vitro chaperone activity of Trx m and Trx f

[283]

trnI/trn A

EDA

PpsbA/TpsbA

2.0 % TSP

The vaccine adjuvant EDA from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts

[284]

trnI/trn A

Proinsulin

PpsbA/TpsbA

47 % TSP in tobacco, 53 % TLP in lettuce

Oral delivery of proinsulin in plant cells lowered glucose levels comparably to injectable commercial insulin

[285]

trnI/trn A

HSA

psbA/psbA/psbA

~11 % TSP

First report of human blood protein in chloroplasts; function not evaluated

[286]

trnI/trn A

IGF

psbA/psbA/psbA

32.7 % TSP

Promoted growth of cultured HU-3 cells in a dose-dependent manner

[287]

trnI/trnA

FIX

PpsbA/TpsbA

1 mg g−1 DW (0.56 % TLP)

Oral delivery of CTB-FIX lettuce cells suppressed inhibitor formation against FIX in hemophilia B mice

[6]

trnI/trnA

FIX

Ppsba/TpsbA

3.8 % TSP; 0.4 mg g−1 FW

Tolerance induction via complex immune regulation, involving tolerogenic dendritic and T-cell subsets

[288]

trnI/trnA

GAA

Ppsba/TpsbA

5.7 mg g−1 DW

Reduced toxic antibody responses in enzyme replacement therapy in Pompe mice

[289]

trnI/trnA

ACE2

Ang-(1–7)

PpsbA/TpsbA

CTB–ACE2: 2.14 % TLP

CTB-Ang1–7: 8.7 % TLP

Oral delivery of ACE2 and Ang (1–7) significantly improved cardiopulmonary structure and functions, decreased the elevated right ventricular systolic blood pressure and improved pulmonary blood flow in animals with induced pulmonary hypertension

[161]

trnI/trn A

BACE

Prrn/TpsbA

2.0 % TSP

Immunogenic response against the BACE antigen in mice

[290]

trnI/trn A

IFNα2b

Prrn/TpsbA

3 mg g−1 FW

Protected cells against VSV CPE and HIV; increased MHC I antibody on splenocytes and total number of natural killer cells and protected mice from a highly metastatic lung tumor

[291]

trnI/trn A

CTB-pins

Prrn/T7g10/TpsbA and rps16

16 % TSP in tobacco, 72 % TLP in lettuce

CTB-proinsulin-fed non-obese diabetic mice significantly decreased inflammation (insulitis); insulin-producing β cells in pancreatic islets were highly protected, increased in insulin production with lower blood or urine glucose levels; increased expression of immunosuppressive cytokines

[128, 292]

rbcL/accD

IFN-γ

PpsbA/TpsbA

6 % TSP

Protection of human lung carcinoma cells against infection by encephalomyocarditis virus

[293]

rbcL/accD

hTrx

PpsbA/Trps16

1 % TSP

Protected mouse from hydrogen peroxide

[294]

rbcL/accD

A1AT

PpsbA/TrbcL

2 % TSP

Binds to porcine pancreatic elastase

[295]

rbcL/accD

TGFβ3

Prrn/T7g10/psbC

12 % TLP

Inhibits mink lung epithelial cell proliferation

[296]

trnV/3′rps12

hCT-1

Prrn/G10L/Trps16

5 % TSP

Biologically active on human hepatocarcinoma cell line

[297]

trnV/rps7/12

hST

PpsbA or Prrn/G10L/Trps16

0.2–7.0 % TSP

Promotes growth of Nb2 cells in a dose-dependent manner

[298]

trnfM/trnG

pal, cpl-1

Prrn/T7g10/TpsbA

~30 % TSP

Bacteriolytic activity and kills Streptococcus pneumoniae, the causative agent of pneumonia

[299]

trnI/trn A

ESAT-6

5′psbA/3′psbA

~7.5 % TSP

Hemolysis of red blood cells and GM1 binding

[165]

trnI/trn A

AMA1

5′psbA/3′psbA

7.3 % TSP in tobacco, 13.2 % TSP in lettuce

Long-term immunity against cholera challenge; inhibition of malarial parasite; protection correlated with IgA and IgG1

[164]

trnI/trn A

MSP1

5′psbA/3′psbA

10.1 % TSP in tobacco, 6.1 % TSP in lettuce

trnI/trn A

2 L21

5′psbA/3′psbA

6.0 % TSP

trnI/trn A

Pag

5′psb/3′psbA

~29.6 % TSP

Macrophage lysis assay, systemic immune response, toxin neutralization assay, mice survived (100 %) challenge with lethal doses of anthrax toxin

[300, 301]

trnI/trn A

L1

PpsbA/TpsbA

20–26 % TSP

Induced systemic immune response and produced neutralizing antibodies in mice

[302]

trnI/trnA

RA4

PpsbA/T psbA

0.2 % TLP

Oral administration elicited both mucosal and systemic Th1/Th2 responses to reduce Toxoplasma parasite load

[303]

trnI/trnA

rFaeG

PpsbA/TrbcL

>1 % DW

Transplastomic plants expressing the rFaeG protein could possibly be used for delivery of an oral vaccine against porcine F4+ ETEC infections

[304]

trnI/trn A

F1-V

Prrn/TpsbA

14.8 % TSP

Orally immunized mice heavily challenged with plague (Yersinia pestis) were protected better than those given IP injections

[305]

trnI/trn A

CTB-2 L21

PpsbA/TpsbA

31.1 % TSP

Immunogenic in mice following IP or oral administration

[306]

trnI/trnA

VP8*

psbA/psbA/Trps16

600 μg g−1 FW

Induced strong immune response and virus neutralization

[307]

trnI/trn A

CtxB

Prrn/ggagg/TpsbA

4.1 % TSP

Efficient GM1 ganglioside-binding

[308]

trnI/trn A

LTB

Prrn/ggagg/TpsbA

2.5 % TSP

GM1 ganglioside-binding assay

[309]

trnI/trn A

LecA

Prrn/T7g10/TpsbA

7 % TSP

Systemic immune response in mice

[310]

trnI/trn A

BACE

Prrn/TpsbA

2.0 % TSP

Immunogenic response against the BACE antigen in mice

[290]

rbcL/accD

OspA, OspA-T

PpsbA/TpsbA

1–10 % TSP

Systemic immune response and protection against Borrelia burgdorferi (Lyme disease)

[311]

trnN/trn R

LTB

Prrn/T7g10/TrbcL

2.3 % TSP

GM1 ganglioside-binding assay; oral immunization partially protected mice from cholera toxin challenge

[312]

trnN/trnR

DPT

Prrn/T7g10/TrbcL

0.8 % TSP

Immunogenic in orally inoculated mice with freeze-dried chloroplast-derived multi-epitope DPT protein

[313]

trnN/trnR

C4V3

Prrn/T7g10/TrbcL

~15 μg mg−1 DW

Plant-derived C4V3 has elicited both systemic and mucosal antibody responses in mice, as well as CD4+ T cell proliferation responses

[314]

trnN/trnR

L1

Prrn/TrbcL

>2 % of TSP

Proper folding and display of conformational epitopes for L1 in the fusion protein by antigen capture ELISA

[315]

trnfM/trnG

p24

Prrn/T7g10/TrbcL

~4 % TSP

Induced strong CD4+ and CD8+ T-cell responses in mice

[316]

trnGtrnfM

HEV E2

Prrn/psbA/TpsbA

1.09 ng μg−1 TSP

Immune response in mice against hepatitis E virus

[317]

trnH/trnK

CSFV E2

Prrn/TpsbA

1–2 % TSP

Immune response in mice against swine fever

[318]

rrn16/rps12/7

TetC

Prrn/T7 g10/TrbcL

atpB/TrbcL

10–25 % TSP

Mice developed systemic immune response and survived the tetanus toxin challenge

[319]

rrn16/trnI

E7

PpsbA/Trps

3–8 % TSP

Several therapeutic HPV-specific E7-based vaccine formulations have been tested in animal models and some have advanced into clinical trials

[320]

Abbreviations: Ang (1–7) Angiotensin (1–7), BACE human b-site APP cleaving enzyme, Bgl β-glucosidase, CPE carbapenemase-producing Enterobacteriaceae, CTB cholera toxin B subunit, DGDG digalactosyldiacylglycerol, DPT diphteria, pertussis, tetanus, DW dry weight, EDA extra domain A-fibronectin, ELISA enzyme-linked immunosorbent assay, ETEC enterotoxigenic Escherichia coli, EX4 exendin-4, FVIII coagulation factor VIII, FW fresh weight, HPV human papilloma virus, IP intraperitoneal, MBP myelin basic protein, MGDG monogalactosyldiacylglycerol, NR not recorded, RbcS small subunit of RuBisCO, RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase, TLP total leaf protein, TSP total soluble protein, VSV vesicular stomatitis virus

Impact of sequence diversity in the chloroplast genome on transgene integration

Figure 3a shows examples of transplastomic genomes that have been transformed with either an endogenous or a heterologous flanking sequence. Every single nucleotide change in the heterologous sequence was subsequently edited out and corrected to achieve 100 % homology to the native sequence within the intergenic spacer region (Fig. 3a). The repetitive editing process significantly reduces the efficiency of transgene integration when using heterologous flanking sequences. This challenge is made even more difficult by inadequate conservation of intergenic spacer regions, even within the same family. Figure 3c shows comparisons of 21 of the most variable intergenic spacer regions; only four of the >150 spacer regions, including the trnl/trnA spacer region, are conserved among members of the Solanaceae. Among grass chloroplast genomes, not a single intergenic spacer region is conserved [8]. This necessitates construction of species-specific chloroplast vectors using endogenous flanking sequences and underscores the need to sequence the chloroplast genomes of economically important crop species.

Ideal sites in the chloroplast genome for transgene integration

The selection of a suitable intergenic spacer region from among more than 100 sites found in each chloroplast genome is a major concern. Statements on the lack of positional effects in the transplastomic literature are common and are used to contrast chloroplast genetic engineering with nuclear transgene integration, which is often associated with profound differences in the expression of transgenes dependent on their site of integration. Evidence shows, however, that there are also positional effects within the chloroplast genome (Table 2). IR regions are found in duplicate in most chloroplast genomes; therefore, transgenes should be inserted within the IR region instead of the SSC or LSC regions because this should double the copy number of transgenes. Integration of a transgene cassette into one copy of the IR facilitates integration into the other copy, thereby enhancing selection pressure to achieve homoplasmy through this copy correction mechanism, a characteristic feature of the chloroplast genome [126128]. Therefore, the site of integration plays a crucial role in transgene expression level and in enhancing homoplasmy under selection by antibiotics. Most importantly, in all sequenced chloroplast genomes within a single plant species, the DNA sequence in one copy of the IR is identical to that in the other copy, without any exception (Table 1).

An early controversy in the chloroplast genetic engineering field was the suitability of transcriptionally silent spacer regions, where native genes (for example, rbcL/accD) are located on opposite strands of the chloroplast genome, or transcriptionally active spacer regions, where native genes (for example, trnA/trnI) are located within operons on the same strand. After a herbicide resistance gene was introduced into the transcriptionally active spacer region for the first time [129], most subsequent studies preferentially used this site of integration (Table 2). The integration of transgenes into the transcriptionally active spacer region (trnl/trnA) has led to 25-fold higher expression of transgenes compared with the transcriptionally silent spacer region (rbcl/accD) [130], possibly due to the presence of multiple promoters (heterologous and endogenous) that enhance transcription. Introns present within trnI/trnA genes (used as flanking sequences) also provide efficient processing of native or foreign transcripts. The trnA gene intron includes a chloroplast origin of replication and produces more copies of the template (chloroplast vectors) for integration of the transgene cassette [131]. In fact, among 114 transgenes in different plant species in Table 2, 71 are integrated at the trnA/trnI site of the chloroplast genome, confirming the unique advantages of this site [127, 129, 130].

Role of chloroplast genome regulatory sequences in transgene expression

In addition to the site of integration, regulatory sequences located upstream (promoter, 5′ UTR) and downstream (3′ UTR) of transgenes play a major role in determining their expression level. The psbA regulatory region, first used almost 25 years ago [131], still appears to be the best option for use in an expression cassette, as the psbA gene encodes the most highly translated protein in the chloroplast [132] and it can also mediate light-induced activation of translation [128]. Indeed, almost all highly expressed transgenes (>70 % TSP, >25 % dry weight) utilize the psbA regulatory region; among 114 transgenes expressed via the chloroplast genome, 84 use the psbA regulatory sequence (Table 2). Other endogenous regulatory sequences that are used include rbcL and atpA, which result in lower transgene expression levels than the psbA promoter/5′ UTR.

Using regulatory regions from photosynthetic genes has the advantage of light regulation, making them ideal for transgene expression in photosynthetic organs (leaves; Fig. 3d, e). However, when the lettuce psbA regulatory region was used in tobacco chloroplasts or vice versa, transgene expression was dramatically reduced (Fig. 3d, e) [128]. Nucleotide differences within the psbA 5′ UTR between tobacco and lettuce (Lactuca sativa) resulted in changes that decreased the interaction of RNA-binding proteins and produced variation in the size of the stem, bulge, and terminal loop of the UTR [128]. In addition, most regulatory proteins (including sigma factors that bind to the promoter region) are nuclear encoded and transported to chloroplasts. This underscores a caveat associated with using regulatory sequences for transgene expression: the need to make species-specific chloroplast vectors to accommodate highly specific regulatory region-binding proteins.

Heterologous regulatory sequences are necessary for transgene expression that is independent of cellular control, especially in non-photosynthetic organs such as fruits and edible roots, where chloroplast protein synthesis is poor [133]. A heterologous UTR (T7 gene10) was first evaluated for expression in leaves [127, 134] and was subsequently tested in non-green tissues. When the expression of BETAINE ALDEHYDE DEHYDROGENASE (BADH) was regulated by the T7 gene10 UTR in carrot (Daucus carota) plants, 75 % of the expression level in leaves was observed in non-green edible roots, conferring the highest level of salt tolerance (400 mM NaCl) found in the published literature (Fig. 4i, j) [135]. Although T7 gene10 has been successfully used to engineer salt tolerance in non-green tissues, its expression level is not as high as that of the psbA regulatory sequence in leaves [136]. The only other heterologous UTR that expressed transgenes at high levels is that from the Bacillus thuringiensis (Bt) operon [137]. Use of this operon produced the highest level of insecticidal toxin protein (52 % TLP) ever reported in the published literature [137]. These high levels of toxin accumulation in chloroplasts could result from the combination of high-level expression and protein stability; the Bt protein formed cuboidal crystals within chloroplasts (Fig. 4e) due to co-expression of a chaperone that facilitates folding. When fed, transplastomic leaves, cotton bollworm (Helicoverpa sp.) were killed with a single bite of leaf and insects that had 40,000-fold increased resistance to Bt were also killed (Fig. 4f, g). Nevertheless, expression of this transgene in tomato fruit is very poor [133, 138, 139] and further research is needed to enhance transgene expression in fruits.
Fig. 4

Engineering the chloroplast genome to confer biotic/abiotic stress tolerance or expression of high-value products. ad Industrial production of blood clotting factor IX (FIX) bioencapsulated in lettuce plants in a hydroponic cGMP facility. a Biomass production of FIX-expressing plants. bd Steps in capsule preparation. After harvesting and lyophilization of fresh leaves, freeze-dried FIX-accumulating leaves were powdered and prepared as capsules [6]. eg 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]

Engineering the chloroplast genomes for biotechnology applications

Conferring stress tolerance

In the past decade, chloroplast genetic engineering has focused primarily on the overexpression of target genes with the potential to enhance biotic stress tolerance, which is very important for plant protection and yield enhancement. Yield loss due to insect pests can be very serious in many countries. In addition to cotton bollworm resistance conferred by hyper-expression of Bt protein in chloroplasts [137], there are many other striking recent examples of improved biotic stress tolerance. Retrocyclin-101 and Protegrin-1 protect against Erwinia soft rot and tobacco mosaic virus (TMV), which result in yield loss in several cultivated crops [140]. Whitefly and aphid resistance has been accomplished by expressing β-glucosidase [141], which releases insecticidal sugar esters from hormone conjugates. Multiple resistances against aphids, whiteflies, lepidopteran insects, and bacterial and viral pathogens were achieved by expressing the Pinellia ternata agglutinin (PTA) gene in the chloroplast genome [142]. More than 40 transgenes have been stably integrated into and expressed within the chloroplast genome, conferring important agronomic traits, including insect resistance in edible crops cabbage (Brassica oleracea) [143], soybean [144, 145], and eggplant (Solanum melongena) [146].

More recently, scientists have begun to explore new strategies to downregulate specific target genes. One such approach is to express double-stranded RNAs (dsRNAs) within the chloroplast genome and to use RNA interference (RNAi) to confer the desired agronomic traits, mainly resistance to insects that cause severe yield loss. This strategy has been demonstrated by expressing long or short dsRNAs that activate RNAi and disrupt target genes in insects, providing efficient protection against insects without the need for chemical pesticides. One such example is the suppression of three essential proteins required for insect survival—lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450), and V-ATPase—using dsRNAs in the tobacco chloroplast system [147]. Each dsRNA was expressed independently in chloroplasts and leaves were fed to insects. The transcription level of target genes in Helicoverpa insects decreased to almost undetectable levels in the midgut, resulting in a significant reduction in the net weight of larvae and in pupation rate [147]. Transplastomic potato plants producing β-actin-targeting long dsRNA were lethal to Colorado potato beetle (Leptinotarsa decemlineata) larvae, providing yet another crop protection mechanism [148].

Synthesis of enzymes and biomaterials

In addition to improved resistance against both biotic and abiotic stress, the chloroplast genome has been engineered to produce useful enzymes, biomaterials, and biofuels, or even to enhance biomass. The first report of metabolic engineering using chloroplast genomes produced the highest level of the poly(p-hydroxybenzoic acid (pHBA) polymer (25 % dry weight) in normal healthy plants despite the diversion of a major metabolic intermediate [149]. The first use of plant-derived enzyme cocktails for the production of fermentable sugars from lignocellulosic biomass was accomplished recently [150]. Unlike the single biofuel enzymes previously expressed in chloroplasts, nine different genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts using a new technique that enabled the insertion of fungal genes with several introns, eliminating the need to prepare cDNA libraries. Industrial fermentation systems are currently limited by high cost and low production capacity; chloroplast-derived enzyme cocktails offer several striking advantages, including significantly reduced cost, improved stability of chloroplast-derived enzymes, and no need for enzyme purification. Interestingly, expression of β-glucosidase released hormones from conjugates, resulting in elevated phytohormone levels and increased biomass [141], an unexpected outcome of enzyme expression.

Enhancing nutrition

Seed oils, such as those from soybean, rapeseed (Brassica napus), and maize, are the major dietary source of vitamin E. They have very low α-tocopherol content but relatively high levels of γ-tocopherol. Only a few seed oils, such as sunflower (Helianthus annuus) seed oil, contain high levels of α-tocopherol, an important precursor of vitamin E [151]. γ-Tocopherol is the biosynthetic precursor of α-tocopherol, suggesting that the α-tocopherol biosynthetic pathway catalyzed by γ-tocopherol methyl transferase (γ-TMT) is the rate-limiting step [152]. Engineering of the γ-tmt gene into the chloroplast genome resulted in the formation of multiple layers of the inner chloroplast envelope (Fig. 4h) due to γ-TMT overexpression, with around tenfold higher conversion of γ-tocopherol to α-tocopherol in seeds [153]. Likewise, introducing lycopene β-cyclase genes into the tomato plastid genome increased the conversion of lycopene into provitamin A (β-carotene), with obvious phenotypic changes (Fig. 4k) [154].

Biopharmaceuticals

At present, protein drugs are extremely expensive; for example, >90 % of the global population cannot afford insulin, a drug needed to treat the global diabetes epidemic. The high cost of protein drugs is due to their production in prohibitively expensive fermentation systems (which cost more than $450–700 million to build depending on their capacity [155, 156]), prohibitively expensive purification from host proteins, the need for refrigerated storage and transport, and the short shelf-life of the final product. Protein drugs made by plant chloroplasts overcome most of these challenges because they do not require expensive fermentation systems and are produced in federal drug administration (FDA)-approved hydroponic greenhouses (Fig. 4a) [157]. Lettuce leaves expressing protein drugs are lyophilized and stored indefinitely at ambient temperature without losing their efficacy (Fig. 4b–d) [6]. The plant cell wall protects protein drugs from acids and enzymes in the stomach because human enzymes do not digest plant cell wall glycans. Human gut microbes, however, have evolved to break down every glycosidic bond in the plant cell wall and therefore release the protein drug into the gut lumen, directing its delivery to the blood or immune system [158, 159].

Oral delivery of several human therapeutic proteins expressed in chloroplasts is highly efficacious in the treatment of several human diseases, including diabetes, cardiovascular disease, pulmonary hypertension, and Alzheimer’s disease. Most proteins were expressed in tobacco chloroplasts for initial evaluation and were subsequently expressed in lettuce chloroplasts for advancing them to the clinic. Oral delivery of exendin-4, which modulates the secretion of insulin in a glucose-dependent manner, lowered glucose in diabetic animals by stimulating the production of insulin in a manner similar to that of the injectable drug [160]. Oral delivery of angiotensin-converting enzyme 2 (ACE2) and angiotensin (Ang) (1–7) significantly improved cardiopulmonary structure and function, decreased elevated right ventricular systolic blood pressure, and improved pulmonary blood flow in animals with induced pulmonary hypertension [161]. Oral delivery of plant cells expressing ACE2 and Ang (1–7) also reduced endotoxin-induced uveitis (EIU) and dramatically decreased cellular infiltration and retinal vasculitis, as well as damage and folding in experimental autoimmune uveoretinitis [158]. It is also possible to orally deliver protein drugs across the blood–brain barrier to the Alzheimer’s brain to remove plaques [162].

The first industrial-scale production of human blood clotting factor in a cGMP facility was reported recently [6] (Fig. 4a–d). In a 1000 ft2 hydroponic cGMP facility, it is possible to produce up to 30,000 doses for a 20-kg pediatric patient. Clotting factor made in lettuce was stable for up to 2 years when lyophilized cells were stored at ambient temperature, completely eliminating the need for the cold chain. This enables the first commercial development of an oral drug and addresses the extremely expensive purification, cold storage and transportation, and short shelf-life of current protein drugs. Oral delivery of a broad dose range was effective in the prevention of antibody formation after injection of clotting factor IX (FIX), further facilitating human clinical studies.

Vaccines against infectious diseases

The current iteration of vaccines, using attenuated bacteria or viruses, offer protection against major infectious diseases but they also present major challenges. For example, the oral polio vaccine that is used around the globe has caused severe polio resulting from mutations and recombination with other viruses [163]. In addition, all current vaccines require cold storage and transportation, making distribution in developing countries a major challenge. Many of these challenges can be overcome by using chloroplasts.

One successful chloroplast-derived vaccine conferred dual immunity against cholera and malaria in animal studies [164]. Cholera is a major disease causing high mortality, with the only licensed vaccine being not only expensive but also limited in its duration of protection. No vaccine is currently available for malaria. The cholera toxin-B subunit (CTB) of Vibrio cholerae was fused to the malarial vaccine antigen apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) and expressed in lettuce or tobacco chloroplasts. While no suitable models exist to test human malaria, a cholera toxin challenge using mice immunized with chloroplast-expressed CTB was highly effective and provided the longest duration of protection in the published literature [164]. These early results show that chloroplasts are ideal for producing low-cost booster vaccines against several infectious diseases [165] for which the global population has been primed previously (Table 2), but lack of an oral priming strategy is still a major limitation in this field.

Moving forward

It is amazing that the chloroplast genome can express >120 foreign genes from different organisms, including bacteria, viruses, fungi, animals, and humans. The insertion of commercially useful traits, including herbicide and insect resistance, into soybean resulted in high-level expression and superior transgene containment, with no antibiotic selectable markers; but even so, these lines were not developed commercially. Nevertheless, recurring concerns about insect resistance against biopesticides have resulted in new USDA requirements on planting Bt corn [122], which may eventually require utilization of the transplastomic approach to confer agronomic traits. The nuclear transgenic approach is inadequate to develop products when higher-level transgene expression is a requirement. Thus, chloroplast transformation has a unique advantage in advancing the field of molecular farming for the production of vaccines, biopharmaceuticals, or other bio-products.

Although products with high-level expression have now advanced to the clinic or are in commercial development, a better understanding of chloroplast translation is required to improve several other gene products. The availability of chloroplast genome sequences should help in the development of codon optimization programs using highly expressed chloroplast genes, but among the ~3000 cultivated crops, sequenced chloroplast genomes are available for crops from fewer than 70 genera. Major funding agencies have not supported crop chloroplast genome sequence projects because of the misconception that all chloroplast genomes are similar, as evidenced by the publication of fewer than ten crop chloroplast genome sequences between 1986 and 2004. This review illustrates the importance of sequencing more crop chloroplast genomes for various biotechnology applications. Furthermore, new selectable markers are needed to transform the chloroplast genomes of cereals, which has been elusive for the past two decades.

Chloroplast genome sequences will be valuable assets in herbal medicine. Most medicinal plants are rare species and very little information is available to confirm their identity. DNA barcodes derived from chloroplast genomes will be useful for identifying varieties and resources; this concept is also valuable in the identification of the origin of cultivated crops and their close relatives to enhance breeding or transfer of useful traits. Molecular techniques to sequence the genomes of single chloroplasts could help to eliminate chloroplast-like sequences that are present in the mitochondrial or nuclear genome. The ability to sequence chloroplast genomes using minimal leaf materials could help us to understand variations in different segments of a variegated leaf in horticultural crops. Further, determining complete chloroplast genome sequences from fossils or recently extinct plants could shed more light on chloroplast genome evolution; help us to understand these species’ inadequate fitness to cope with environmental changes; and help us to build new phylogenetic trees. The technology for isolating DNA from fossils is already available [166168]. All of these goals can be accomplished with less expensive and more accurate genome sequences, utilizing longer read sequencing technology and new bioinformatics tools.

Abbreviations

ACE2, Angiotensin-converting enzyme 2; BAC, bacterial artificial chromosome; Bt, Bacillus thuringiensis; CTB, Cholera toxin B subunit; cGMP, Current Good Manufacturing Processes; CTB, Cholera toxin B subunit; dsRNA, double-stranded RNA; FIX, clotting factor IX; infA, translation initiation factor 1; IR, inverted repeat; LSC, large single copy; ndh, NAD(P)H dehydrogenase; NCBI, National Center for Biotechnology Information; NGS, next-generation sequencing; RNAi, RNA interference; SSC, small single copy; γ-TMT, γ-Tocopherol methyltransferase; TSP, total soluble protein; UTR, untranslated region; Ang (1–7), Angiotensin (1–7)

Declarations

Acknowledgements

We acknowledge valuable contributions from editors of Genome Biology (Drs Dominique Morneau and Ripudaman Bains) to enhance the flow and presentation to non-specialists and reviewers for their critical comments.

Funding

Research from the Daniell Laboratory included in this review was supported by the Bill and Melinda Gates Foundation (OPP1031406), the National Institutes of Health (NIH; R01 HL107904, HL109442, GM 63879, EY 024564), Novo Nordisk, Bayer, and Department of Energy ARPA-E grants to Henry Daniell.

Authors’ contributions

HD and CSL wrote this review. MY assembled Table 2 and Figs. 3 and 4 with guidance from HD. WJC assembled Table 1 and Figs. 1 and 2 with guidance from CSL. All authors read and approved the final manuscript.

Competing interests

Henry Daniell, as a pioneer in the field of chloroplast genetic engineering, has several patents in this field but has no financial conflict of interest to declare. A complete list of published patents is available at the Google Scholar weblink: http://scholar.google.com/citations?user=7sow4jwAAAAJ&hl=en

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biochemistry, School of Dental Medicine, University of Pennsylvania
(2)
Agricultural Biotechnology Research Center, Academia Sinica

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