key: cord-1045761-3resp4dm
authors: Daniell, Henry; Jin, Shuangxia; Zhu, Xin‐Guang; Gitzendanner, Matthew A.; Soltis, Douglas E.; Soltis, Pamela S.
title: Green giant—a tiny chloroplast genome with mighty power to produce high‐value proteins: history and phylogeny
date: 2021-02-22
journal: Plant Biotechnol J
DOI: 10.1111/pbi.13556
sha: 9d26ca55e5e27aab25875a79a070eaf230e45b45
doc_id: 1045761
cord_uid: 3resp4dm

Free‐living cyanobacteria were entrapped by eukaryotic cells ~2 billion years ago, ultimately giving rise to chloroplasts. After a century of debate, the presence of chloroplast DNA was demonstrated in the 1960s. The first chloroplast genomes were sequenced in the 1980s, followed by ~100 vegetable, fruit, cereal, beverage, oil and starch/sugar crop chloroplast genomes in the past three decades. Foreign genes were expressed in isolated chloroplasts or intact plant cells in the late 1980s and stably integrated into chloroplast genomes, with typically maternal inheritance shown in the 1990s. Since then, chloroplast genomes conferred the highest reported levels of tolerance or resistance to biotic or abiotic stress. Although launching products with agronomic traits in important crops using this concept has been elusive, commercial products developed include enzymes used in everyday life from processing fruit juice, to enhancing water absorption of cotton fibre or removal of stains as laundry detergents and in dye removal in the textile industry. Plastid genome sequences have revealed the framework of green plant phylogeny as well as the intricate history of plastid genome transfer events to other eukaryotes. Discordant historical signals among plastid genes suggest possible variable constraints across the plastome and further understanding and mitigation of these constraints may yield new opportunities for bioengineering. In this review, we trace the evolutionary history of chloroplasts, status of autonomy and recent advances in products developed for everyday use or those advanced to the clinic, including treatment of COVID‐19 patients and SARS‐CoV‐2 vaccine.

Chloroplasts have sustained life on this planet for one billion years by providing carbohydrates, amino acids, lipids for human nutrition and O 2 to breathe through the process of photosynthesis. Chloroplasts synthesize the most abundant protein on Earth (RuBisCO) and utilize CO 2 , thereby playing a key role in reducing atmospheric CO 2, and moderating global warming. Although non-Mendelian inheritance was recognized in early 1900s (Baur, 1909; Correns, 1909) , direct evidence for chloroplast DNA was not established until 1960s (Kirk, 1963; Ris and Plaut, 1962) . In the past three decades, chloroplast genomes have been genetically modified to introduce agronomic traits mostly in model systems or to produce therapeutic products or enzymes used in everyday life. The typical maternal inheritance (with rare paternal or biparental inheritance) of chloroplast genomes offers gene containment when foreign genes are integrated into chloroplast genomes. The large copy number of chloroplast genomes (up to 10,000 in each plant cell) offers high levels of expression of introduced foreign genes. Site-specific integration of foreign genes reduces the number of transgenic lines to be evaluated. In addition, the lack of gene silencing or position effects in chloroplast genomes offers uniform expression of foreign genes. Thus, modern-day chloroplasts could play additional major roles in human health and well-being well beyond providing food, feed, fuel and oxygen.

Integration of foreign genes (>300) into chloroplast genomes resulted in their highest levels of expression and conferred the highest level of tolerance or resistance reported to date. Insulin was expressed in chloroplasts up to 70% of leaf protein or higher than RuBisCO (Ruhlman et al., 2010) , and a high-value polymer, pHBA, was expressed up to 26.5% of leaf dry weight (Viitanen et al., 2004) . The Cry2A operon produced folded Bt crystals within chloroplasts (DeCosa et al., 2001) . Chloroplast expression of Tic40 triggered the nucleus to make all inner membrane proteins, resulting in 8-20 layers of the inner envelope membrane (Singh et al., 2008) . Although these are early examples of using very small human proteins or genes of bacterial origin, most of the challenging problems with engineered plastids remained unaddressed until recently. Failure to adequately express human or viral genes in chloroplasts has been addressed recently by developing new algorithms that relied on a large number of sequenced chloroplast genomes (Kwon et al., 2016; Kwon et al., 2018) . When the largest human blood protein (FVIII, 185 kDa monomer, with pentamer assembly) was expressed in the chloroplast, the level reached industry expectations for commercial scale production and clinical evaluation (Kwon et al., 2018) . Codon optimised expression of angiotensin-converting enzyme 2 not only helped to treat pulmonary hypertension, but has also now been tested in the clinic to treat COVID-19 patients (Daniell, 2020) , as discussed in depth below. Codon-optimized expression of a novel human insulin-like growth factor in lettuce chloroplasts promoted musculoskeletal cell proliferation, differentiation and diabetic bone fracture healing (Park et al., 2020) . The expression level of enzymes in chloroplasts and the efficacy exceeded current products made in fermentation systems using almost all evaluation criteria, without any need for purification (Daniell et al., 2019c; Kumari et al., 2019) . Most importantly, a selectable antibiotic resistance gene has been removed from transplastomic chloroplast genomes in edible crops (Daniell et al., 2019b; Kumari et al., 2019; Park et al., 2020) . Production of protein drugs in cGMP facilities, long-term stability of drugs in freeze-dried plant cells (Daniell, 2020; Daniell et al., 2019b; Daniell et al., 2019c; Park et al., 2020; Su et al., 2015) , evaluation of drug substance by toxicology and pharmacokinetic studies (Daniell et al., 2019c) and recent FDA approval of PDs orally delivered peanut cells to treat allergy (Tilles and Petroni, 2018; Vickery et al., 2018) all represent major recent advances in this field. Based on these recent advances, the next decade should be particularly promising for the common use of proteins made in chloroplasts to enhance human health and well-being.

This review offers a brief overview of the history, changes in chloroplast autonomy, the use of plastid DNA sequence data to reconstruct plant evolutionary history, as well as recent advances and future use of chloroplast genomes for biotechnology applications. This summary is not intended to provide a complete list of all chloroplast genomes sequenced or foreign genes expressed but our goal is to focus on a few selected products advanced beyond laboratories for daily use or in clinical development. In addition, philosophical questions on the loss of chloroplast genes during evolution and status of chloroplast autonomy are discussed.

In 1909, Baur and Correns described the revolutionary concept of non-Mendelian inheritance factors located outside the nucleus in the protoplasm (Baur, 1909; Correns, 1909;  Table 1 ). In crosses between variegated, yellow and green Mirabilis jalapa plants, branches always gave rise to the same colour seedlings, without any Mendelian segregation (Correns, 1909) . Studies in the following decades showed different mechanisms for exclusion of male chloroplast DNA during sexual fusion. The generative cells divide unequally during pollen formation and do not receive any chloroplasts. In angiosperms, each generative cell forms two sperm cells, one of which fuses with a female gamete to form a zygote and the other of which unites with polar nuclei to produce endosperm, that is the process referred to as 'double fertilization'. In this process, male chloroplasts or DNA is specifically excluded or degraded, assuring maternal inheritance of the chloroplast genome in both the vast majority of angiosperms and other land plants as well, with a few noteworthy exceptions (Daniell, 2002; Daniell, 2007; Hagemann and Schroeder, 1989) .

Role of maternal inheritance of chloroplast genomes gained greater attention when foreign genes were introduced into chloroplast genomes. Transgene escape via pollen and the possibility of weedy relatives capturing this valuable trait have been a major concern in nuclear transgenic crops. Therefore, integration of the herbicide resistance gene via the chloroplast genome and maternal inheritance of transgenes was considered a major accomplishment, and this invention was featured on the cover of Nature Biotechnology (Daniell et al., 1998) . Subsequently, maternal inheritance of numerous foreign genes integrated into the chloroplast genome has been documented (Daniell et al., 2016a; Daniell et al., 2016b;  Table 1 ).

In addition to transgene containment via maternal inheritance, chloroplast engineering produces products in leaves, facilitating their harvest before appearance of any reproductive structures, thus offering complete containment. Therefore, field tests of products were conducted a decade ago for biopharmaceuticals expressed in chloroplasts (Arlen et al., 2007) (Table 1) . More recently, USDA-APHIS certifies that 'transplastomic lines do not fit the definition of a regulated article under USDA-APHIS regulations 7 CFR part 340, because there are no plant pest components' (Kwon and Daniell, 2015) . These advantages should facilitate field production of products engineered via the chloroplast genome.

The concept of cyanobacteria as evolutionary precursors of chloroplasts was proposed a century ago (Mereschkowsky, 1905) , and a cyanobacterial origin of all plastids is now well established (Delwiche et al., 1995) . About 1.5-2 billion years ago, free-living cyanobacteria were entrapped by early eukaryotic cells. This entrapment process has been experimentally demonstrated in laboratories through the uptake of algal chloroplasts by plant protoplasts or of green chloroplasts by albino protoplasts, and regeneration of green or variegated plants (Bonnett, 1976; Bonnett and Eriksson, 1974) . Subsequent to this ancient entrapment event, there was a massive transfer of cyanobacterial genes to the nuclear genome over the long course of evolutionary history of green plants. In Arabidopsis, 18% of the nuclear genes originated from the ancestral plastid genome (Martin et al., 2002) . Therefore, most of the plastid proteins are nuclearencoded and genetically dependent on the host cell, and chloroplasts are no longer obligate endosymbionts but organelles (Cavalier-Smith, 1985) .

There has been an enormous reduction in gene content from the ancestral cyanobacteria to the plastid genomes found in photosynthetic eukaryotes. The genome of the cyanobacterium Synechocystis PCC 6803 is 3,573 kbp and contains~3,200 genes, and the plastid genome in the red alga Porphya purpurea is only 191 kbp, with~250 genes (Reith and Munholland, 1995) . Plastids of red algae and glaucocystophytes encode genes for several biosynthetic pathways, nitrogen assimilation and metabolic regulation, in addition to genes involved in protein synthesis and photosynthesis (Delwiche, 1999 Baur (1909) , Correns (1909) 1929 'Plastome' as hereditary factor Renner (1929) 1962

Chlamydomonas Cp DNAvisual evidence Ris and Plaut (1962) 1963

Broadbean cp DNA-visual evidence Kirk (1963) , Leff et al (1963 Leff et al ( ) 1974 Leff et al ( -1976 Uptake of isolated chloroplasts by protoplasts and plant regeneration Bonnett and Eriksson (1974) , Bonnett (1976 Bonnett ( ) 1983 Bonnett ( -1986 Synthesis of thylakoids, macro-grana functional PSI, PSII in isolated chloroplasts Daniell et al. (1983 Daniell et al. ( , 1986 1986

Complete sequence of the first plant chloroplast genome (tobacco) Shinozaki et al. (1986 Shinozaki et al. ( ) 1987 First foreign genes (chloramphenicol acetyltransferase, B-lactamase) expressed in isolated chloroplasts - McFadden (1987) 1990 First chloroplast vectors using the psbA regulatory sequences subsequently used in large majority of transgene expression studies in chloroplasts Daniell et al. (1990 Daniell et al. ( , 1991 1990

First foreign gene expression in plant chloroplasts (chloramphenicol acetyl transferase, Bglucuronidase) using the gun powder or helium gene gunthe most reproducible gene delivery system for chloroplast transformation Daniell et al. (1990 Daniell et al. ( , 1991 , Ye et al. (1990) 1991

The aadA gene as chloroplast selectable marker-subsequently used in most transgenes integrated into chloroplast genomes. Goldschmidt-Clermont (1991) 1993

The aadA gene integration into the tobacco chloroplast genome in the large single-copy region Svab and Maliga (1993) 1995 Cry1Ac gene expression in chloroplasts to confer resistance to insects, followed by subsequent reports in other genes biosynthesis of the peptidoglycan wall, and this species retains a cyanobacterial cell wall (L€ offelhardt and Bohnert, 1994) . Loss of the peptidoglycan wall during the evolution of land plants is a significant irreversible step towards reducing plastid survival outside the plant cell. Reduction of plastid genome size continues (up to 113 kbp) with loss of both the inverted repeat region and ndh genes in land plants (Sanderson et al., 2015) . Most land plant plastid genomes contain 110-130 genes, with~80 genes coding for proteins involved in photosynthesis and other processes. In parasitic plants, photosynthetic genes are lost, but the chloroplast genomes retain genes essential for protein synthesis and origin of DNA replication (Banerjee and Stefanovi c, 2019). Despite the heavy loss of genes from the ancestral cyanobacterial genome, isolated chloroplasts still retain the ability to perform protein synthesis because they contain > 50% of the total ribosomal complement of photosynthetic cells, DNA, DNA polymerase, RNA polymerase and tRNAs (Ellis, 1977) . Use of S 35 methionine and specific protein synthesis inhibitors for chloroplast (chloramphenicol) and cytosolic (cycloheximide) ribosomes made it possible to distinguish proteins synthesized in each compartment (Ellis, 1977) . Isolated intact chloroplasts were shown to synthesize proteins, capable of photosystem I activity with cyclic phosphorylation (Daniell et al., 1983) and form macrograna (Rebeiz et al., 1984) with photosystem II activities Daniell and Sarojini, 1984) during greening in vitro. It is remarkable that protein synthetic capacity is retained in parasitic plants when all photosynthetic genes were lost.

Renner coined the term 'plastome' to describe plastid hereditary factors (Renner, 1929 (Renner, , 1934 (Table 1) . Although the presence of DNA within plastids was debated for decades, Ris and Plaut (1962) were the first to convincingly demonstrate the presence of DNA in Chlamydomonas chloroplasts using electron micrographs with 25 A microfibril size, sensitivity to nuclease digestion and Feulgen reaction and yellow-green fluorescence of acridine orange staining. Subsequently, DNA was shown in broad bean (Kirk, 1963) and other chloroplasts and was referred to as 'satellite DNA' (Leff et al., 1963) (Table 1) .

Two decades after discovery of organellar DNA, the complete chloroplast genome of the first chloroplast genome (tobacco) was published by Shinozaki et al in 1986 (Table 1) . Only a few crop chloroplast genome sequences were published in the following decade, and this paucity of data was a major limitation in engineering crop chloroplast genomes. The misconception was that chloroplast genome sequences are highly conserved among crop chloroplast genomes. However,~50% of the chloroplast genome contains coding sequences which are highly conserved, but the intergenic sequences that are essential for transgene integration or that contain regulatory sequences are not conserved. Among species of Solanaceae, only four of > 150 intergenic sequences are conserved, including the trnI/trnA spacer region used in our chloroplast vectors . Not even a single spacer region is conserved among sequenced grass (2011) chloroplast genomes (Saski et al., 2007) . Transgene cassettes are introduced into the chloroplast genome intergenic spacer regions, using native genes to facilitate homologous recombination. However, when tobacco chloroplast genome flanking sequences were inserted into the lettuce chloroplast genome, unique nucleotides were completely eliminated or modified to achieve 100% homologous recombination, dramatically reducing transformation efficiency (Ruhlman et al., 2010) . Likewise, when tobacco psbA regulatory sequences (promoter, 5', 3' UTR) were used in lettuce chloroplasts, there was 80-97% reduction in translation when compared to the endogenous regulatory sequences (Ruhlman et al., 2010) . As a result, species-specific chloroplast vectors with endogenous genes and regulatory sequences are required for efficient foreign gene expression. Based on the work noted above, in the last decade, significant efforts were made to sequence crop chloroplast genomes that are used in everyday life including soybean (Saski et al., 2005) , other legumes , potato , tomato , grape , coffee (Samson et al., 2007) , cotton , orange (Bausher et al., 2006) , cassava , carrot (Ruhlman et al., 2006) and cereals (Saski et al., 2007) . For a current list of edible crop chloroplast genomes, readers are referred to FAO: http:// www.fao.org/fileadmin/templates/ess/documents/world_census_ of_agriculture/appendix3_r7.pdf. -and Table 2 . As discussed below, chloroplast genome sequences facilitate codon optimization and offer the best regulatory sequences to enhance translation and transgene integration. Understanding the origins of economically important cultivated species facilitates breeding and prevents cross-contamination of plants used in herbal medicine. Moreover, an understanding of the diversity of chloroplast genomes, in terms of both structure and sequence, is important for developing efficient systems for genetic engineering. However, among~3,000 cultivated crops, completely sequenced chloroplast genomes are available for fewer than 70 genera. Among these, <80 complete chloroplast genomes are available in the NCBI database. However, the One Thousand Plants Transcriptome Project (1KP; onekp.com), as well as other recent efforts, have contributed over 1,000 complete or nearly complete plastid genomes to global databases, most of these from plants that are not of economic importance (Gitzendanner et al., 2018; Leebens-Mack, 2019; Li et al., 2019) ; hence, our understanding of plastid genomes across the Tree of Life has improved dramatically in the past decade.

For a number of reasons (abundance, single-copy genes, lack of recombination and appropriate rate of nucleotide evolution), the plastid genome has long been the primary workhorse for studies of plant phylogeny and evolution. The size and structure of the plastid genome have been remarkably conserved across land plant evolution (although intergenic spacer regions and regulatory sequences are not well conserved), in stark contrast to the enormous variation in size and structure of the plant mitochondrial genome, and this conservation has facilitated the use of both sequence data and plastome rearrangements in phylogenetic analyses. As noted above, transfer of genes from the plastome to the nuclear genome has reduced the size of the plastid genome over the course of green plant evolution, with chlorophytes having larger plastid genomes and more genes than streptophytes, particularly land plants. There is also evidence of some plastid gene movement to the mitochondrial genome.

Phylogenetic analyses using plastid genes have been conducted across a range of divergences from the species level and to very deep levels. Particularly at deeper levels (e.g. at divergences traditionally recognized at the family level and deeper), plastid data have been of enormous value. Initial studies employed only rbcL (encoding the large subunit of RuBisCO); in a landmark study showing the utility of plastid gene sequences, a collaboration of 43 investigators provided the first DNA phylogenetic framework for seed plants based on an analysis of 499 species (Chase et al., 1993) . Most recently, next-generation sequencing has enabled the sequencing of the complete plastid genome and the assembly of large phylogenetic trees across all green plants (Gitzendanner et al., 2018; Ruhfel et al., 2014) ; other studies of plastid loci have focused on major subclades of green plants . Plastid phylogenetics ushered in the most the fundamental changes in our understanding of plant relationships in the past 150 years, revealing the major clades of green plants, the sister group to land plants, relationships across land plants, with a major reshaping of our understanding of moss, liverwort, fern, gymnosperms and angiosperm phylogeny. Not only have these studies resulted in a clearer understanding of evolutionary relationships, they have also prompted major new classifications for the angiosperms (APG IV 2016) and ferns (Pteridophyte.Phylogeny.Group, 2016); these are groundbreaking classifications that represent dramatic changes from anything previously published based on morphology.

In some cases, the plastid genome has exhibited enough variation to be of utility in studies at the population level and also in phylogeographic analyses (Brunsfeld et al., 2001; Soltis et al., 1997) , although not on the scale observed with mtDNA in animals. Although hybridization has long been known to be a major force in plant evolution, molecular studies using plastid genes have revealed many unsuspected past hybridization events showing that hybridization is even more prevalent in plants than thought, with hundreds of documented cases of introgression of plastid genomes. Most of our current framework of green plant phylogenetic relationships is based on plastid genome sequence data, and current classifications are largely based on plastid gene phylogenetics. Only in the past few years as nuclear gene sequencing has become more routine have comparable nuclear gene topologies been generated. Importantly, there are discordances between plastid and nuclear trees, not only at shallow levels where introgression has long been detected, but also at deep levels , indicating putative ancient reticulation.

Studies of plastid genes and genomes have also revealed the complex history of the plastid green plant clade, with secondary and tertiary endosymbiotic events (representing the capture of photosynthetic green or red algae) occurred in other lineages, including brown algae, red algae and Euglena (Keeling, 2004; Keeling, 2010; Palmer et al., 2004) . Together, this increasingly large set of plastid genes and genomes from across green plant phylogeny and other clades of photosynthetic eukaryotes provides the sequence information and resources, not only for tracing plant evolution, but also for chloroplast genetic engineering.

The technical innovations (Moore et al., 2006; Stull et al., 2013; Uribe-Convers et al., 2014) that enabled use of the entire plastome, or at least most of the~80 protein-coding genes, as well as the four tRNA genes, typical of an angiosperm plastome, in phylogenetic analyses (Gitzendanner et Stull et al., 2015) continue to be instructive about the evolution of the genome itself. Despite long-standing debate about if, when and how to combine data from different sources (molecules and morphology) or different genomes (nuclear and organellar), combining all plastid genes in a single analysis, perhaps with different evolutionary parameter values, has generally received consensus, as these genes are linked and represent a single chromosome (perhaps viewed as a single character, Doyle, 1992) , and most studies have indeed combined plastid genes into a single analysis. Recent evidence, however, has revealed extensive topological discordance among trees built from individual plastid genes, both across angiosperms as a whole (Walker et al., 2019) and within legumes . Although differences among plastid gene trees may arise due to biological causes-for example heteroplasmic recombination and gene transfer among plastid, nuclear and mitochondrial genomes -strong conflict is unexpected, particularly given that heteroplasmy is considered rare (Sancho et al., 2018; Sullivan et al., 2017) , limiting the possibility of recombination. The factors responsible for observed discordant plastid gene trees are not well understood on an empirical level, and more research is needed into heteroplasmy and intergenomic transfer, although results to date point to both stochastic and systematic error, the latter arising due to misspecifications of the evolutionary model used in the phylogenetic analysis (Walker et al., 2019) . Regardless of the cause of gene tree conflict, plastid genes, if combined into a single analysis, may conflate multiple phylogenetic signals, muddying the overall inference of both topology and branch lengths, with consequences for downstream analyses of divergence times, diversification, character evolution and more; thus, greater exploration of plastid gene trees is needed in future studies.

At the dawn of the GMO revolution in the 1980s, introducing herbicide resistance genes into the nuclear genome, using Agrobacterium-mediated transformation (Shah et al., 1986) with the possibility of escape of transgenes via pollen, was publicly debated. Another major concern was the development of resistance in insects to bacterial insecticidal (Bt) proteins produced in plants at low expression levels via the nuclear genome. Therefore, the concept of chloroplast genetic engineering was first demonstrated in isolated chloroplasts (Daniell et al., 1991; Daniell and McFadden, 1987; Daniell et al., 1983; Rebeiz et al., 1984) , with the goal of reintroduction of chloroplasts into protoplasts to regenerate transplastomic lines (Bonnett, 1976; Bonnett and Eriksson, 1974) (Table 1) . However, invention of the gene gun by John Sanford at Cornell University eliminated the need for the latter step and facilitated introduction of foreign DNA directly into chloroplasts of plant cells (Daniell, 1993; Daniell et al., 1990; Ye et al., 1990) ( Table 1) . After 30 years of research on foreign gene expression in chloroplasts, >75% of foreign genes (Daniell et al., 2016a; Daniell et al., 2016b) use the psbA regulatory sequences used in this first report . Although several methods for DNA delivery into chloroplasts have been reported, gene gun delivery is still the only reproducible method (Table 1) . Goldschmidt-Clermont (1991) introduced the first selectable marker gene for chloroplasts-the aadA gene (Table 1) , which was subsequently used for transforming the tobacco chloroplast genome (Svab and Maliga, 1993) and most other crops . Although foreign genes were introduced into the transcriptionally silent spacer region (Svab and Maliga, 1993) , transcriptionally active spacer region in the inverted repeat region of the chloroplast genome was proposed by Daniell et al (1998) and is now the most widely used spacer region for transgene integration (Daniell et al., 2016a, b; Daniell et al., 1998; .

Chloroplast genomes have been engineered to express foreign genes from bacterial, fungal, protozoan or human genomes for various biotechnology applications. It is quite astounding that a bacterial operon from Bacillus thuringiensis was expressed utilizing bacterial regulatory sequences and achieved the highest level of Bt protein reported (DeCosa et al., 2001) . Small human genes (insulin) are expressed at very high levels (up to 70% of leaf total protein (Boyhan and Daniell, 2011; Ruhlman et al., 2010) ). However, when large human genes are introduced into the chloroplast genome, there are poorly translated. Therefore, a new codon optimization program was developed based on codon usage of the most highly expressed psbA genes from 133 sequenced chloroplast genomes (Kwon et al., 2016) . The first iteration used a new codon optimizer algorithm that followed the codon usage hierarchy observed among sequenced psbA genes. Synonymous codons for each amino acid were ranked according to their frequency of use. Codon optimization of the human blood clotting factor FVIII-HC (2262 bp) modified 406 codons out of 754 amino acids. Native FVIII-HC used the CTC leucine codon 11 times but the codon-optimized HC eliminated all CTC codons. Likewise, another rare codon, TCA (serine), used 16 times in the native FVIII-HC coding sequence, was completely eliminated after codon optimization. Collectively, the codon-optimization algorithm eliminated 105 rare codons from the FVIII-HC native human gene, resulting in enhanced expression. Similar codon optimization of the CTB-FVIII-LC gene resulted in successful expression of the largest human blood protein (FVIII, 185 kDa monomer, with pentamer assembly) in chloroplasts (Kwon et al., 2018) , and this has now advanced to commercial production and clinical trials. Codon-optimized Ace2 is now tested in the clinic to treat COVID-19 patients (Daniell, 2020) .

The global enzyme market was valued at US $11 billion in 2018; it is used in the food/beverage (37%), cleaning (26%), bioenergy (15%) and animal feed (13%) industries. Rising demand in detergents, textile and wastewater treatment applications are propelling significant growth in the enzyme industry. Enzymes are now used as biofertilizers and soil enhancers to accelerate growth through enhancing beneficial microbes. Carbohydrase led the product segment, with 48% market share in 2018. Globally, the Asia Pacific region leads the industrial enzyme demand ($4.6 billion), followed by Europe ($2.68 billion) and the United States has the largest (38%) market share of industrial enzymes. Waxes and pectins decrease water absorption of native cotton fibres; contrary to common understanding, natural cotton fibre does not absorb water or dyes (Lin and Hsieh, 2001) . To enhance absorption of harsh chemicals, alkaline pH and other severe conditions have been used, but these are environmentally problematic in that they release toxic effluents. Therefore, pectinases are used for textile bioscouring to enhance water absorbency of cotton fibres, without causing cellulose destruction. Likewise, lipases are used in the detergent industry to remove oils or butter stains (Jaeger and Reetz, 1998) . However, performance of current commercial lipases is limited by minimal activities in alkaline pH or at higher temperature (detergents are alkaline and washing machines often use hot water).

Mannans are found in chocolate, ice cream and tomato ketchup, which are unfortunately the most common cloth stains (Chauhan et al., 2012) . Mannanase hydrolyses insoluble mannan into water-soluble smaller oligosaccharides (Dhawan and Kaur, 2007) , thereby facilitating stain removal. However, current mannanase products are not active through a wide range of temperatures or pH (Sarmiento et al., 2015) . Mannanase is also used in the paper and pulp industries, bioethanol production, oil and gas well stimulation, food and feed, nutraceutical and pharmaceutical industries (Srivastava and Kapoor, 2017; Zyl et al., 2010) . Endoglucanases and exoglucanases are useful in enhancing colour brightness and fabric softening (Agrawal, 2017) . These enzymes are used in denim biowashing, biostoning and biopolishing to remove dye from the fibril surface with minimal damage to fabrics (Anish et al., 2007; Ara ujo et al., 2009; Miettinen-Oinonen and Suominen, 2002) . Cocktails of cellulases, hemicellulases and pectinases are used for fruit juice clarification, concentration and reducing viscosity (Brito and Vaillant, 2012; Sharma et al., 2016) .

Currently, industrial enzymes are produced in microbial systems that are expensive and that rely on decades-old processes. Building fermentation facilities and their maintenance free of contamination is the first challenge. Purification of enzymes from host cells and their formulation to increase concentration, stability and cold storage/transportation are all very expensive. Therefore, several enzyme products made in leaves and expressed in chloroplasts (Agrawal et al., 2011; Jin et al., 2011; Leelavathi et al, 2003; Verma et al., 2010) were launched recently by PhylloZyme in order to replace prohibitively expensive microbial enzyme processes (Daniell et al., 2019a; Daniell et al., 2019c; Kumari et al., 2019) . This is the first report of commercially launched protein products made in leaves (Table 1 ; Figure 1 ). Leaf pectinases, endoglucanases, lipases or mannanases were validated with 23 commercial microbial enzyme products for textile (dye binding, removal, depilling), detergent (stain removal) or juice industry (clarification) applications. Most leaf enzymes function in broad pH/temperature ranges as crude leaf extracts without the need for purification, and leaf enzymes could be stored as lyophilized plant cells at ambient temperature for several months/years, without loss of enzyme activity.

Commercially produced enzymes are widely used in daily life from coffee/juice in the morning, textiles worn during the day (enhancing water absorption, dye binding), cleaning of dishes/clothes (detergents), evening beverages (wine, beer) or digesting food (fat, carbohydrates). Currently, these enzymes are produced in yeast, fungi or bacteria in fermentation systems. Life cycle analysis of b-glucosidase, used for digesting milk products, revealed that, for each kg of b-glucosidase enzyme, 52 kg of CO 2 are released from fermentation, along with 18,140 kg wastewater and > 15 kg of solid debris and carbon monoxide, methane and other toxic gasses, in addition to environmental stress caused by toxic chemicals used in cleaning the fermenters used in enzyme production (Feijoo et al., 2017) . Considering the millions of tons of microbial biomass produced every day, dealing with global CO 2 emission and toxic effluents is a major environmental challenge.

Here, we use cellulase production with the tobacco enzyme production platform to evaluate the carbon capturing potential of leaf enzyme technology. Assuming a standard tobacco production for 12 weeks, reaching a final leaf area index (LAI) of 2.5, and average photosynthetic properties as reported in (Schmidt et al., 2019) , respiration of 30% of the total photosynthetic CO 2 uptake (Amthor, 1989; Amthor, 2000) , then the total CO 2 uptake by such a tobacco canopy to produce 1 kg cellulase will be about 570 kg. If at the end of the growth cycle, if LAI is 6 instead, a net photosynthetic CO 2 uptake of 484 kg is predicted during the production of 1 kg cellulase. Replacement of enzymes made by fermentation by leaf enzymes could capture up to 570 kg CO 2 in addition to preventing 110 kg CO 2 release for a net capture of 680 kg CO 2 for each kg of enzyme/protein produced, altogether leading to decreased anthropogenic greenhouse gas emissions.

Affordable protein drugs and vaccines-from laboratory to the clinic Although protein drugs like insulin save millions of lives, they are not affordable for a large majority of the global population. Indeed, insulin pumps cost $6,000-$12,000 but one third of the global population earn <$2 per day. Therefore, new approaches are needed to produce and deliver protein drugs (PDs) more cost effectively. Edible plant cells offer the opportunity to express and orally deliver PDs. Upon oral delivery, PDs are protected in the stomach from digestion by enzymes because human or animal enzymes do not break beta linkage of plant cell wall polymers. However, when intact plant cells reach the gut, enzymes released by commensal microbes in the gut digest plant cell wall, releasing PDs into the gut lumen (Kumar et al., 2020) . When tags are fused to PDs, they cross gut epithelium and reach the circulatory or immune system (Figure 2) . Therefore, oral delivery of PDs Comparison of chloroplast and microbial enzymes on dye or stain removal, water absorption or juice clarification: (a) Endoglucanase expressed in chloroplasts (a3) removed dye from denim fabric uniformly unlike uneven removal using microbial enzyme (a2) when compared to untreated control (a1). (b): Chloroplast pectinase (b2, b3) clarified orange juice pulp similar to microbial pectinase (b4) when compared to untreated control (b1). (c) Chloroplast pectinase (c4) increased water absorption of cotton fabric similar to microbial enzyme (c3), when compared to controls with water (c1) or untransformed plant extract (c2). (d) Chloroplast mannanase (d4) removed cholate stain more efficiently than microbial enzyme (d3) when compared to untreated control (d1) or with detergent alone (d2). Antibiotic resistance gene was removed from lettuce transplastomic lines but not from tobacco. For further details, see or Kumari et al, 2019 Figure 2 Oral delivery of protein drugs made in lettuce chloroplasts: (a) Production of Ace2/Ang1-7 in lettuce chloroplasts, freeze drying and oral delivery of capsules. (b) Protection of intact plant cells expressing GFP from stomach acids and enzymes and delivery to the gut. Gut microbes release enzymes to digest the plant cell wall and release GFP to gut lumen, which is taken up by gut epithelial cells. (c) SARS-CoV-2 binds to the human Ace2 receptor via the spike protein (target of several vaccines), lowers Ace 2 levels and causes lung/heart injury through inflammation by disregulation of the RAS pathway. (d) Oral delivery of angiotensin-converting enzyme 2 and angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension and reduces right ventricular systolic pressure, right ventricular hypertrophy. Fibrosis and pulmonary vessel wall thickness, which are the symptoms observed in COVID-19 patients. For further details, see Daniell (2020) . Recent advances include expression of PDs in marker-free chloroplast genomes by removal of the antibiotic resistance gene (Park et al., 2020; Daniell et al 2020) , production of lettuce expressing PDs in cGMP facilities (Daniell et al., 2019a Park et al., 2020) , evaluation of drug substance by toxicology and pharmacokinetic studies and recent FDA approval of PDs orally delivered in peanut cells to treat allergy (Tilles and Petroni, 2018; Vickery et al., 2018) . Two major examples are discussed below in the context of developing SARS-CoV-2 vaccine or treating acute/lethal lung/heart failure of COVID-19 patients (Figure 2 ).

There is an urgent need to develop new vaccine strategies for SARS-CoV-2. There are > 50 vaccine clinical trials currently in progress, most of them using the same antigen (spike protein) but produced using DNA, RNA or recombinant proteins (Amanat and Krammer, 2020) . Because all of these are injectable vaccines (except Vaxart), they will primarily produce systemic immunity with suboptimal induction of mucosal surface immunity required to protect at viral entry points (Sui and Berzofsky, 2020) . Induction of mucosal immunity will likely require oral vaccination or antigen delivery via other mucosal routes. In addition, the induction of suboptimal and/or short-lived responses (as seen already in repeat COVID-19 infection) including in the elderly would require additional boosters. In this context, recent development of a polio oral booster vaccine using lettuce chloroplasts, in collaboration with FDA and CDC laboratories, funded by the Gates foundation is highly significant. Oral delivery of codonoptimized polio viral protein 1 common to all polio viral serotypes generated both IgA and IgG1 specific antibodies and conferred protection against all three polio virus serotypes Xiao and Daniell, 2017; Daniell et al, 2019b) . While a single-dose injectable polio vaccine (IPV) did not produce significant levels of IgA and IgG1 titres decreased quickly, oral boosters with VP1 expressed in chloroplasts maintained both IgA and IgG1 up to 400 days. In case of Sabin 1 and Sabin 2, a single dose of IPV resulted in poor seropositivity (<20% or < 40%), but showed~70% seropositivity against Sabin 3. Plant cell boosting showed higher seropositivity against Sabin 1, Sabin 2 and Sabin 3 (80-100% protection). When OPV2 was withdrawn by the World Health Organization because it could revert to virulence by point mutations or recombination with other enteroviruses, most developing countries could not afford a second dose of IPV. In a very similar situation, an affordable oral booster strategy is now developed for SARS-CoV-2 oral booster vaccination, especially to develop prolonged mucosal immunity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), infects host cells via the angiotensin-converting enzyme 2 (ACE2) receptor, resulting in vasoconstriction, hypercoagulability, myocardial/lung injury, fibrosis, inflammation associated with ACE2 down-regulation and/or cytokine storm (Du et al., 2020; Zoufaly et al., 2020) . In healthy people, ACE2 is expressed in type II alveolar lung epithelial cells that produce surfactants and this protects alveoli from collapsing. Most importantly, ACE2 produces anti-inflammatory, cytoprotective angiotensin 1-7 (Ang 1-7) peptide via cleavage of the vasoconstrictor angiotensin II (AngII). Of potential therapeutic relevance, oral delivery of both ACE2 and Ang1-7 bioencapsulated in plant cells significantly decreased right ventricular systolic pressure and improved pulmonary blood flow and right ventricle function in diseased hypertensive animals (Daniell, 2020; Shenoy et al., 2014 ; Figure 2 ). Conceivably, therapeutic delivery of ACE2/Ang1-7 could also be employed to restore a more favourable balance of Ang II and Ang 1-7 in patients with COVID-19 disease. In contrast to exogenously delivered truncated (transmembrane deleted) soluble ACE2 (Zoufaly et al., 2020) , full-length ACE2 accumulates in the lungs at 10-fold higher concentrations than in the plasma, with no evidence of toxicity . Based on these observations, evaluation of therapeutic efficacy and safety of supplementing ACE2 and Ang (1-7) with this existing product in non-critically ill COVID-19 patients in the hospital and at home is in progress. Investigational New Drug application has been filed and has gone through two rounds of FDA review, by employing an integrated Phases 1 and 2 clinical trial design. This clinical trial is planned to treat a growing number of COVID-19 patients to protect them from lung/extrapulmonary injury and heart failure.

Sequenced chloroplast genomes are available for fewer than 70 genera of the~3,000 species of cultivated crops and of fewer than 2,000 of the~350,000 species of flowering plants; thus, further efforts are needed to increase the number of sequenced plastomes. Approximately half of every chloroplast genome contains intergenic spacer regions and regulatory sequences that are not conserved, but essential for chloroplast genetic engineering. The lack of intergenic spacer sequences is an important unmet need. In addition, several attempts to transform cereal chloroplast genomes have been unsuccessful, and advances in this area are needed to apply this concept to confer desired agronomic traits. Almost all successful chloroplast genetic engineering reported here used the gene gun and the aadA gene. Therefore, future investigations could focus on development of new selectable markers and DNA delivery methods to achieve plastid transformation in cereal crops.

Plastid DNA sequences will continue to be an important tool in elucidating relationships and evolutionary processes across not only green plants, but also other photosynthetic lineages of life. Of particular interest in the next decade will be more studies that compare patterns of nuclear and plastid phylogenetic relationships. It is anticipated that many more discordant patterns of relationships will be uncovered across the photosynthetic tree of life, providing novel insights into a deep and recurring history of reticulate evolution. Moreover, understanding the nature of the discordance in the phylogenetic signal of different plastomeencoded genes will yield further insight into the evolutionary constraints on the plastome and thus the utility of different regions for bioengineering.

Chloroplasts play important roles in sustaining life on this planet by providing carbohydrates, amino acids and lipids as sources of animal (including human) nutrition and O 2 , which supports animal life and moderates global warming by trapping CO 2 . In the past two decades, chloroplasts have been called upon to produce proteins (enzymes) used in everyday life and to reduce the cost of drugs to offer affordable health care. Major advances have been made in this area in the past few years. When clinical trials currently in progress are successful, an affordable protein drug production and delivery will be launched soon using chloroplasts as bioreactors. Recent advances augur well for chloroplasts to play a central role in both medicine and in sustaining life on Earth, replacing decades-old prohibitively expensive fermentation and drug delivery technologies. 

Most of the recent publications on chloroplast biotechnology in the Daniell laboratory reviewed here were supported by funding from NIH grant R01 HL 107904, R01 HL 109442 and R01 HL 133191.

Henry Daniell discloses conflict of interest due to several patents on chloroplast genetic engineering and technology founder of PhylloZyme that launched chloroplast enzyme products and biopharmaceuticals research supported in the past by Bayer, Novo Nordisk, Johnson & Johnson and currently by Shire/Takeda.

HD conceived this topic and wrote this review. SJ contributed to sections on crop chloroplast genomes and contents of tables. X-GZ contributed the section on CO 2 emission by fermentation and capture by chloroplasts. MAG, DES and PSS contributed sections on chloroplast genome and phylogenetics.

Bio-stoning of denim-an environmental-friendly approach

Expression of Trichoderma reesei b-mannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis

SARS-CoV-2 Vaccines: Status Report. Immunity

Respiration and crop productivity

The McCree-de wit-penning de vries-thornley respiration paradigms: 30 years later

Application of cellulases from an alkalothermophilic Thermomonospora sp. in biopolishing of denims

Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion

Application of enzymes for textile fibres processing

Field production and functional evaluation of chloroplastderived interferon-alpha2b

Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts

Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

Caught in action: fine-scale plastome evolution in the parasitic plants of Cuscuta section Ceratophorae (Convolvulaceae)

Das Wesen und die Erblichkeitsverh€ altnisse der "Varietates albomarginatae Hort" von Pelargonium zonale

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms

On the mechanism of the uptake of Vaucheria chloroplasts by carrot protoplasts treated with polyethylene glycol

Transfer of algal chloroplasts into protoplasts of higher plants

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide

Enzymatic liquefaction of cell-walls from kent and tommy atkins mango fruits

Comparative phylogeography of northwestern North America: a synthesis

(1985) Protozoa as hosts for endosymbiosis and the conversion of symbionts into organelles

Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth

Cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes

Phylogenetics of seed plants: An analysis of nucleotide sequences from the plastid gene rbcL

Mannanases: microbial sources, production, properties and potential biotechnological applications

Identification and phylogenetic analysis of the complete chloroplast genomes of three ephedra herbs containing ephedrine

Characterization of complete chloroplast genome and phylogenetic analysis of sweet cherry Cerasus avium (L.) Moench

The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae species identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence

Vererbungsversuche mit blass(gelb)gr€ unen und buntbl€ attrigen Sippen bei Mirabilis jalapa

Comparative and phylogenetic analyses of ginger (Zingiber officinale) in the family zingiberaceae based on the complete chloroplast genome

Foreign gene expression in chloroplasts of higher plants mediated by tungsten particle bombardment

Molecular strategies for gene containment in transgenic crops

Transgene containment by maternal inheritance: effective or elusive?

From conception to COVID-19: an arduous journey of tribulations of racism and triumphs

The Authors

Vaccination via chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious human diseases

Containment of herbicide resistance through genetic engineering of the chloroplast genome

Transient expression of bglucuronidase in different cellular compartments following biolistic delivery of foreign DNA into wheat leaves and calli

Radioisotopic evidence for the polypeptides associated with photosystem II

Plant cell-made protein antigens for induction of Oral tolerance

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

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

Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts

Cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Successful in vitro synthesis of oxygen evolving photosynthetic membranes: A milestone in photosynthesis research

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors

The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi

Tracing the thread of plastid diversity through the tapestry of life

Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

Microbial mannanases: an overview of production and applications

Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants

The complete chloroplast genome sequence of Prunus sibirica

A comparative analysis of whole plastid genomes from the apiales: expansion and contraction of the inverted repeat, mitochondrial to plastid transfer of DNA, and identification of highly divergent noncoding regions

Gene trees and species trees: molecular systematics as onecharacter taxonomy

COVID-19: the role of excessive cytokine release and potential ACE2 down-regulation in promoting hypercoagulable state associated with severe illness

Generation and characterization of soybean and markerfree tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance

Generation of fertile transplastomic soybean

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

Protein synthesis by isolated chloroplasts

Complete plastid genome sequence of Vaccinium macrocarpon:structure, gene content, and rearrangements revealed by next generation sequencing

Life cycle assessment of b-Galactosidase enzyme production

A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

Plastid phylogenomic analysis of green plants: A billion years of evolutionary history

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas

Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice

Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts

Rapid evolutionary change of common bean (Phaseolus vulgaris L) plastome, and the genomic diversification of legume chloroplasts

Activation of human mast cells by retrocyclin and protegrin highlight their immunomodulatory and antimicrobial properties

The cytological basis of plastid inheritance in angiosperms

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids

Length dependent accumulation of double stranded RNAs in plastids affect RNA interference efficiency in the colorado potato beetle

Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Complete chloroplast genome sequence of rapeseed (Brassica napus L.) and its evolutionary implications

Complete sequence and comparative analysis of the chloroplast genome of coconut palm (Cocos nucifera)

Complete chloroplast genome sequences of four Allium species: comparative and phylogenetic analyses

Phytoremediation of mercury and organomercurials in chloroplast transgenic plants: Enhanced root uptake, translocation to shoots, and volatilization

Removal of antibiotic resistance genes from transgenic tobacco plastids

Microbial lipases form versatile tools for biotechnology

Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure, gene localization, and characterization of a large inversion

): evidence for at least two independent transfers of rpl22 to the nucleus

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.)

Expression of c-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species

The engineered chloroplast genome just got smarter

Release of hormones from conjugates: chloroplast expression of b-glucosidase results in elevated phytohormone levels associated with significant increase in biomass and protection from aphids or whiteflies conferred by sucrose esters

Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation

Complete sequencing and comparative analyses of the pepper (Capsicum annuum L.) plastome revealed high frequency of tandem repeats and large insertion/deletions on pepper plastome

The complete chloroplast genome sequence of Coix lacryma-jobi L. (Poaceae), a cereal and medicinal crop

Diversity and evolutionary history of plastids and their hosts

The endosymbiotic origin, diversification and fate of plastids

Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng Species

The deoxyribonucleic acid of broad bean chloroplasts

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Plant-based vaccine: mice immunized with chloroplast-derived anthraxprotective antigen survive anthrax lethal toxin challenge

Autoluminiscent plants

Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance

Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology

Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Low-cost oral delivery of protein drugs bioencapsulated in plant cells

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms

The complete chloroplast genome sequence of Zanthoxylum piperitum

Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections

The Authors

One thousand plant transcriptomes and the phylogenomics of green plants

Overproduction of an alkali-and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme

DNA satellites from cells of green and aplastidic algae

The complete chloroplast genome sequence of sugar beet (Beta vulgaris ssp. vulgaris)

Origin of angiosperms and the puzzle of the Jurassic gap

A Phylogenetic Analysis of Chloroplast Genomes Elucidates the Relationships of the Six Economically Important Brassica Species Comprising the Triangle of U

The location and translocation of ndh genes of chloroplast origin in the Orchidaceae family

Direct Scouring of Greige Cotton Fabrics with Proteases

Comparative chloroplast genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale -A wild ancestor of cultivated buckwheat

Carboxysome encapsulation of the CO(2)-fixing enzyme Rubisco in tobacco chloroplasts

The complete chloroplast genome sequence of Safflower (Carthamus tinctorius L

Expression of bar gene in the pastid genome confers herbicide resistance

Complete chloroplast genome sequence of a major economic species, Ziziphus jujuba (Rhamnaceae)

Localized hypermutation and associated gene losses in legume chloroplast genomes

Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing

Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells

Cost-effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies

The complete chloroplast genome of banana (Musa acuminata, Zingiberales): insight into plastid monocotyledon evolution

Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus

Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco

The complete chloroplast genome of an economic plant, Camellia sinensis cultivar Anhua

U¨ber Natur und Ursprung der Chromatoporen im Pflanzenreiche

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus)

The complete chloroplast genome sequence of Fritillaria thunbergii Miq., an important medicinal plant, and identification of DNA markers to authenticate Fritillariae Bulbus

Using plastid genomescale data to resolve enigmatic relationships among basal angiosperms

Rapid and accurate pyrosequencing of angiosperm plastid genomes

Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots

The chloroplast genome of Cerasus humilis: Genomic characterization and phylogenetic analysis

The Complete Chloroplast Genome of Ye-Xing-Ba (Scrophularia dentata; Scrophulariaceae), an Alpine Tibetan Herb

Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

The plant tree of life: an overview and some points of view

The complete chloroplast genome of aniseed tree, Illicium anisatum L. (Schisandraceae)

The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species

Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling

High level expression of a suit of thermostable cell wall degrading enzymes from the chloroplast genome

The Authors

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: its composition and comparative analysis

The complete chloroplast genome sequence of Indian mustard (Brassica juncea L.)

A community-derived classification for extant lycophytes and ferns

Plastome Sequencing of Ten Nonmodel Crop Species Uncovers a Large Insertion of Mitochondrial DNA in Cashew

CHLOROPLAST CULTURE X: THYLAKOID ASSEMBLY IN VITRO

Complete nucleotide sequence of thePorphyra purpurea chloroplast genome

Handbuch f€ ur Vererbungswissenschaft, herausgeg. vonE. BAUR u.M. HARTMANN

Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas

Determination of the melon chloroplast and mitochondrial genome sequences reveals that the largest reported mitochondrial genome in plants contains a significant amount of DNA having a nuclear origin

From algae to angiosperms-inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes

Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in non-obese diabetic mice

Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

Phytoremediation of organomercurial compounds via chloroplast genetic engineering

Plastid phylogenomics of the cool-season grass subfamily: clarification of relationships among early-diverging tribes

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

Comparative plastome genomics and phylogenomics of Brachypodium: flowering time signatures, introgression and recombination in recently diverged ecotypes

Exceptional reduction of the plastid genome of saguaro cactus (Carnegiea gigantea): Loss of the ndh gene suite and inverted repeat

The Linum usitatissimum L. plastome reveals atypical structural evolution, new editing sites, and the phylogenetic position of Linaceae within Malpighiales

Cold and Hot Extremozymes: Industrial Relevance and Current Trends

Complete Chloroplast Genome Sequence of Glycine max and Comparative Analyses with other Legume Genomes

Complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera, and comparative analyses with other grass genomes

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts

The plastid chromosome of spinach (Spinacia oleracea): complete nucleotide sequence and gene organization

The complete chloroplast genome of eggplant (Solanum melongena L.)

The complete chloroplast genome of two Brassica species, Brassica nigra and B. Oleracea

Engineering Herbicide Tolerance in Transgenic Plants

The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth

The complete chloroplast genome sequence of asparagus (Asparagus officinalis L.) and its phylogenetic position within Asparagales

Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

Contradiction between plastid gene transcription and function due to complex posttranscriptional splicing: an exemplary study of ycf15 function and evolution in angiosperms

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression

The genome of woodland strawberry (Fragaria vesca)

Arabidopsis Tic40 expression in tobacco chloroplasts results in massive proliferation of the inner envelope membrane and upregulation of associated proteins

Chloroplast DNA intraspecific phylogeography of plants from the Pacific Northwest of North America

The Authors

The Complete Chloroplast Genomes of Two Lespedeza Species: Insights into Codon Usage Bias, RNA Editing Sites, and Phylogenetic Relationships in Desmodieae (Fabaceae: Papilionoideae). Plants (Basel) 9

Production, properties, and applications of endo-b-mannanases

High-yield production of a human therapeutic protein in tobacco chloroplasts

Resolving basal lamiid phylogeny and the circumscription of Icacinaceae with a plastome-scale data set

A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes

Nuclear phylogenomic analyses of asterids conflict with plastome trees and support novel relationships among major lineages

Complete chloroplast genome sequence of Omani lime (Citrus aurantiifolia) and comparative analysis within the rosids

Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice

Myeloid Cell-Mediated Trained Innate Immunity in Mucosal AIDS Vaccine Development

Interspecific Plastome Recombination Reflects Ancient Reticulate Evolution in Picea (Pinaceae)

Exploring deep incongruence in the angiosperms: an example involving the COM clade

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene

The chloroplast genome sequence of mungbean (Vigna radiata) determined by high-throughput pyrosequencing: structural organization and phylogenetic relationships

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

FDA-approved peanut allergy treatment: The first wave is about to crest

A comparative analysis of the Lactuca and Helianthus (Asteraceae) plastid genomes: identification of divergent regions and categorization of shared repeats

Protection against tetanus toxin using a plant-based vaccine

A long PCRbased approach for DNA enrichment prior to next-generation sequencing for systematic studies

Characterization of the chloroplast genome sequence of oil palm

Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae

Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis

Characterizing gene tree conflict in plastome-inferred phylogenies

The complete chloroplast genome of an irreplaceable dietary and model crop, foxtail millet

Characterization of the complete chloroplast genome of longan (Dimocarpus longan Lour.) using illumina paired-end sequencing

Complete chloroplast genome of the medicinal plant Amomum compactum: gene organization, comparative analysis, and phylogenetic relationships within Zingiberales

Long-term evaluation of mucosal and systemic immunity and protection conferred by different polio booster vaccines

The complete plastid genome of Chinese cinnamon

Comparative Analysis of two Sugarcane Ancestors Saccharum officinarum and S. spontaneum based on Complete Chloroplast Genome Sequences and Photosynthetic Ability in Cold Stress

Analyses of the complete genome and gene expression of chloroplast of sweet potato

The complete chloroplast genome of cultivated apple (Malus domestica Cv

Characterization of the complete chloroplast genome of the Chinese sorghum, Sorghum bicolor from China

Sequencing and Structural Analysis of the Complete Chloroplast Genome of the Medicinal Plant Lycium chinense Mill

The complete chloroplast genome sequence of Chicory

The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.)

The complete chloroplast genome of Torilis scabra (Apiaceae)

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Optimization of delivery of foreign DNA into higher-plant chloroplasts

Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco

Echinochloa chloroplast genomes: insights into the evolution and taxonomic identification of two weedy species

Complete chloroplast genome sequences of important oilseed crop Sesamum indicum L. PLoS One 7, e35872. ª 2021 The Authors

Development of chloroplast genome resources for peanut (Arachis hypogaea L.) and other species of Arachis

Cas9/gRNAmediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Complete chloroplast genome sequences of Rehmannia chingii, an endemic and endangered herb

Generation of virus-resistant potato plants by RNA genome targeting

Pest control. Full crop protection from an insect pest by expression of long doublestranded RNAs in plastids

The complete plastid genome sequence of Panax notoginseng, a famous traditional Chinese medicinal plant of the family Araliaceae

Exploration of Plastid Phylogenomic Conflict Yields New Insights into the Deep Relationships of Leguminosae

The complete chloroplast genome sequence of the Cucurbita pepo L. (Cucurbitaceae)

The complete sequence of chloroplast genome from mango (Mangifera indica var GuiFei)

The complete chloroplast genome sequence of the Apium graveolens L. (Apiaceae)

Fungal bmannanases: Mannan hydrolysis, heterologous production and biotechnological applications

The Authors