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REVIEW
Challenges and perspectives in commercializing plastid
transformation technology
Niaz Ahmad1, *, Franck Michoux2, Andreas G. Lössl3, and Peter J. Nixon4
1 Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic
Engineering, Jhang Road, Faisalabad, Pakistan
2 Alkion Biopharma SAS, 4 rue Pierre Fontaine, 91058 Evry, France
3 Department of Applied Plant Sciences and Plant Biotechnology, University of Natural
Resources and Applied Life Sciences (BOKU), Vienna, Austria
4 Department of Life Sciences, Sir Ernst Chain Building–Wolfson Laboratories, Imperial
College, South Kensington Campus, London SW7 2AZ, UK
* Correspondence: [email protected]
Received 30 July 2016; Accepted 6 September 2016
Running head: Developments in plastid transformation technology
Editor: Christine Raines, University of Essex
Running head: Ahmad et al.
Highlight
Transformation of the plastid genome has emerged as an alternative tool to develop
transgenic plants. This review discusses recent developments and the current challenges.
Abstract
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Plastid transformation has emerged as an alternative platform to generate transgenic
plants. Attractive features of this technology include specific integration of transgenes—
either individually or as operons—into the plastid genome through homologous
recombination, the potential for high-level protein expression, and transgene containment
because of the maternal inheritance of plastids. Several issues associated with nuclear
transformation such as gene silencing, variable gene expression due to the Mendelian
laws of inheritance, and epigenetic regulation have not been observed in the plastid
genome. Plastid transformation has been successfully used for the production of
therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various
agronomic traits including resistance to biotic and abiotic stresses. However, these
demonstrations have usually focused on model systems such as tobacco, and the
technology per se has not yet reached the market. Technical factors limiting this
technology include the lack of efficient protocols for the transformation of cereals, poor
transgene expression in non-green plastids, a limited number of selection markers, and
the lengthy procedures required to recover fully segregated plants. This article discusses
the technology of transforming the plastid genome, the positive and negative features
compared with nuclear transformation, and the current challenges that need to be
addressed for successful commercialization.
Abbreviations: CCM, carbon concentrating mechanism; GFP, green fluorescent protein;
IR, inverted repeat; LSC, large single copy; PTM, post-translational modification; SSC,
small single copy; TIB, temporary immersion bioreactor; TSP, total soluble proteins.
Key words: Plastids, transformation of plastid genome, site-specific integration of
transgenes, gene containment, metabolic engineering, molecular farming
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Introduction
Plastids are a group of organelles present in the cells of plants, algae, and some protists
that are essential for viability (Waters and Pyke, 2005). It is now widely accepted that
plastids originated around ~1.5 billion years ago when a cyanobacterial cell was engulfed
by a heterotrophic eukaryote (Bock, 2015). Over the years, the host and the incoming
bacterial cell developed a symbiotic relationship, which involved: (i) synchronization of
cell division of the cyanobacterium with that of the host cell, (ii) extensive transfer of the
incoming cell’s genetic material to the host cell nucleus and/or deletion of redundant
genetic information, (iii) evolution of mechanisms to import proteins back into the
cyanobacterium from the host cell, and (iv) the development of signalling pathways for
efficient coordination of gene expression in the plastid and in the host nucleus. The result
of this massive gene transfer to the host cell nucleus has been a drastic reduction in the
size of the plastid genome (termed plastome) of modern-day plants compared with the
genomes of free-living cyanobacteria. Analysis of the Arabidopsis chloroplast proteome
has revealed the presence of ~3000 proteins (Jarvis, 2004; Kleffmann et al., 2004), of
which only 87 are encoded on the plastome (Martin et al., 2002).
The sequence, structure, organization, and mechanisms of protein expression all
clearly indicate that the current-day plastome is a remnant of an earlier cyanobacterial-
like genome. A comparative analysis of the circular plastome of higher plants shows that
many features such as overall size, gene density, ‘AT’ content, and the presence of
inverted repeats (IR), which bifurcates the plastome into large single copy (LSC) and
small single copy (SSC) regions, are highly conserved between species (Carbonell-
Caballero et al., 2015; Wicke et al., 2011). The IR regions are mirror images of each
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other, and are maintained with respect to each other by an intrinsic mechanism known as
copy correction (Khan et al., 2007).
Another interesting feature of plastids is their diversity in function. Modern-day
plants differentiate to produce more than 50 different types of cell (Pyke, 2009). The
incoming plastids have likewise been manipulated during their evolutionary journey to
carry out a range of different functions according to the type of cell/tissue in which they
reside. Therefore, in addition to photosynthesis, plastids are involved in an array of
cellular processes, including starch metabolism, sulphur metabolism, fatty acid
biosynthesis, nitrogen assimilation, and the synthesis of various amino acids (Pyke, 2009;
Van Dingenen et al., 2016; Warzecha, 2016). The study of plastid biology is therefore
essential for a holistic view of plant physiology.
The plastid genome also provides an alternative site for the stable insertion of
transgenes in plants and is also often considered an ‘environmentally friendly’ form of
plant genetic engineering, as plastid DNA (in most crops) is largely excluded from
pollen. Chloroplast transformation was first demonstrated for the unicellular green alga
Chlamydomonas reinhardtii (Boynton et al., 1988) followed by the higher plants tobacco
(Svab et al., 1990a) and tomato (Ruf et al., 2001). Since then, the chloroplast genome has
been manipulated to produce vaccine antigens, commercial enzymes, hormones,
antibodies, pharmaceuticals, and biomaterials, and engineered to provide various
agronomic traits, including resistance against various biotic (viral, fungal, and bacterial
diseases) and abiotic (e.g. salt, drought, heavy metals) factors (see Table 1). However,
the number of crop plants in which chloroplast transformation has been reported remains
disappointingly low (see Table 2) and the technology has not yet reached the field. In this
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article, we will describe the process of transforming the plastid genome and its salient
advantages, followed by the challenges hampering its successful implementation to field
crops.
Transforming the plastid genome: the state of the art
One of the intriguing features of the plastome is its incredibly high copy number,
reaching levels of about 10 000 copies per mesophyll cell (Shaver et al., 2006). This high
copy number is thought to produce and maintain the photosynthetic apparatus during
plant development (Jarvis and López-Juez, 2013) and also plays an important role in
plastome stability by eliminating deleterious mutations through homologous
recombination (Garton et al., 2007; Jarvis and López-Juez, 2013).
The current procedure for experimentally transforming the plastome starts with
the construction of a transforming plasmid containing an antibiotic-resistance cassette
that functions in chloroplasts and a separate expression cassette containing the gene or
genes of interest to be expressed. Flanking these cassettes are two plastome fragments,
typically 1–2 kb in size, termed flanking regions or targeting regions (Fig. 1), which help
integrate foreign DNA at a specific location in the plastome through homologous
recombination. The plastome-transforming plasmids are usually Escherichia coli
plasmids that are unable to replicate inside the plastid. These constructs are delivered to
plastids through a gene-gun-mediated particle delivery system after coating the DNA on
to the surface of microparticles (0.4–1.0 µm) of gold or tungsten (Maliga and
Tungsuchat-Huang, 2014). Other methods such as polyethylene glycol-mediated
transformation or microinjection can also be used to deliver foreign DNA into plastids,
but the transformation and regeneration efficiency of these methods is relatively low
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compared with biolistic DNA delivery (Day and Goldschmidt‐Clermont, 2011; Maliga,
2002). Subsequently, antibiotic resistance selects for transgenes that have been stably
integrated into the plastome (Maliga, 2002, 2003). The most widely used selectable
marker is the bacterial aadA gene, expressed using plastid expression regulatory
elements, which encodes the enzyme aminoglycoside 3′′-adenylyltransferase, which
detoxifies spectinomycin and streptomycin. It is a positive dominant selection marker and
has been reported to give high transformation efficiency due to selective amplification of
transformed copies of plastid DNA (Svab et al., 1990b; Svab and Maliga, 1993).
When the DNA is delivered into plastids, only a few copies of the plastome are
initially transformed, resulting in a so-called heteroplasmic state. Homoplasmy, where all
copies of the plastome contain the transgene, is achieved by subculturing the bombarded
explant in vitro under selection (Maliga and Tungsuchat-Huang, 2014). Under these
conditions, both the plastid and the plastome copy number decrease from several hundred
to much fewer. The change in overall plastid copy number is thought to allow plastids
carrying the selectable marker to divide at a much faster rate than wild-type versions
under the stringent selection conditions used (Maliga and Bock, 2011). This dilution
cycle is repeated under constant selection pressure until wild-type copies of the plastome
are lost. Once homoplasmy is achieved, the marker gene can be excised to generate
marker-free plants. Several systems are available, such as Cre/loxP, Int (integrase) phage
recombinase system, or intrinsic homologous recombination employing flanking direct
repeats (see Day and Goldschmidt‐Clermont, 2011 for a comprehensive review).
Key advantages of chloroplast transformation technology
Natural gene containment
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One of the major concerns over the cultivation of transgenic plants in the field is the
outcrossing of transgenes and their dissemination into wild species. This perceived risk
stems from the observation that plants exchange their genetic material with their relatives
through pollen-mediated introgression (Stewart et al., 2003). For example, genes
encoding resistance against three herbicides—Roundup, Liberty, and Pursuit—have been
transferred from nuclear transformants of canola (Brassica napus L.) to weeds (Steward,
2000). Containment of transgenes has therefore become an important consideration when
releasing genetically modified plants into the field. In this context, transforming non-
nuclear genomes, such as those in the plastid and mitochondrion, may provide a
mechanism for natural gene containment due to their maternal mode of inheritance (Jaffe
et al., 2008). For instance, inheritance of plastids in the majority of angiosperms is
predominantly maternal (Greiner et al., 2015; Hagemann, 2004), which means that the
transgene inserted into the plastid genome of these species should in principle not be
dispersed via pollen. However, containment is not absolute, and transmission of
transgenic plastids to pollen has been measured to be in the range of 0.00024–0.008% in
tobacco (Ruf et al., 2007; Svab and Maliga, 2007) and 0.0039% in Arabidopsis thaliana
(Azhagiri and Maliga, 2007). In the case where gene containment needs to be absolute,
new methods have been developed based on in vitro propagation of transplastomic
biomass (see Transgene containment in chloroplast genome is not absolute, below, for
more details). The plastid DNA can also be transferred to the nuclear genome (Huang et
al., 2003; Sheppard et al., 2008; Wang et al., 2012), which raises the possibility that
transgenes originally inserted into the plastome could be spread like a classic nuclear
transformant (Allainguillaume et al., 2009; Gilbert, 2013).
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Site-specific integration of the transgene into the plastome
As illustrated in Fig. 1, site-specific integration of a gene of interest at a predetermined
location in the plastid genome is advantageous as it avoids several issues such as gene
silencing and unwanted mutations resulting from the random integration of transgenes.
Although homologous recombination has also been reported for nuclear genomes (Vieler
et al., 2012), the standard procedure involving Agrobacterium transformation often leads
to random insertion of transgenes (Kohli et al., 2010). The foreign gene may also interact
with native nuclear genes and, as a result of this non-allelic interaction, the function of
native genes could be masked or vice versa (Scheid et al., 1991). Such potential
challenges, which are inevitable in the case of nuclear transformation (Qin et al., 2003),
have not been observed when foreign genes are introduced into the chloroplast genome
(Bock, 2015; Maliga and Bock, 2011).
Besides the insertion of foreign genes, chloroplast transformation has also been a
useful tool to probe the role of plastid-encoded proteins through the creation of knockout
and site-directed mutants (Bock, 2015).
Transformation of the chloroplast genome often yields high
expression levels
The much higher copy number displayed by the plastome favours high expression of
foreign proteins in comparison to nuclear expression. For example, expression of the β-
subunit of enterotoxigenic E. coli (LT-B) from the tobacco nuclear genome was less than
0.01% of total soluble protein (TSP) (Haq et al., 1995), whereas transformation of the
chloroplast led to a 410-fold increase in expression of the same protein (Daniell et al.,
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2001a). There are now several examples of extremely high expression of foreign proteins
in tobacco chloroplasts, with levels exceeding 70% TSP (Table 1).
Expressing and confining a foreign protein to the chloroplast might also be useful
in protecting plants from the toxic effects of that protein. For example, the presence of
even small levels of cholera β-toxin in the tobacco cytosol (0.3% TSP) resulted in stunted
plant growth (Arakawa et al., 1997), whereas chloroplast transformants expressing 14-
fold higher levels were not adversely affected (Daniell et al., 2001b). Proteins that are
difficult to produce in other systems, such as antibiotics and cell-wall-degrading
enzymes, have also been successfully expressed in chloroplasts (Espinoza-Sánchez et al.,
2016; Oey et al., 2009a, 2009b; Petersen and Bock, 2011; Verma et al., 2010a) (Table 1).
However, there are examples where overexpression of foreign proteins can affect
chloroplast function and plant fitness (Scotti and Cardi, 2014). For example, expression
of tetanus toxin fragment C (TetC) at 25% TSP, but not at 10% TSP, proved detrimental
to the growth of host plants (Tregoning et al., 2003). Similarly, expression of glutathione-
S-transferase (GST) in tobacco chloroplasts at ~7% TSP induced cytoplasmic male
sterility (Ahmad et al., 2012a), whereas no such effect was observed at lower levels of
accumulation (Le Martret et al., 2011).
Gene expression in chloroplasts is highly regulated at the transcriptional,
translational, and post-translational levels (Maliga, 2003). Therefore, the accumulation of
foreign proteins can be increased or decreased several fold simply through the judicious
use of regulatory elements, such as the promoter and 5′ untranslated regions, and at the
post-translational level by manipulation of the N-terminal coding sequence of the gene of
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interest (Maliga, 2003) and co-expression of a chaperone to aid protein folding (De Cosa
et al., 2001; Lin et al., 2014; Whitney et al., 2015).
Engineering complex metabolic pathways
Metabolic pathways often require the concerted action of multiple enzymes to confer a
desired trait on a plant, such as protection from biotic or abiotic stresses, improvement of
nutritional value, production of metabolites in bulk for industrial applications, or
increasing photosynthesis for improving crop yields (Bock, 2013). For example, the DNA
responsible for encoding the nitrogen-fixation enzymes in Klebsiella pneumoniae is 24
kb long and consists of 20 genes (Arnold et al., 1988), and the polysaccharide formation
gene cluster in Streptococcus is 25 kb long and composed of 16 genes (Cieslewicz et al.,
2001). Introducing these genes via nuclear transformation would require several
transformation events and several backcrosses. One of the prokaryotic characteristics of
the plastid genome is the operonal organization of genes, allowing several genes to be co-
transcribed. This feature opens up the possibility of using chloroplast transformation to
introduce natural and artificial operons into the plastome in a single transformation step.
However, post-transcriptional processes within the chloroplast, such as RNA editing,
removal of introns, processing of mRNA ends, and cleavage of larger mRNA transcripts
(reviewed by Bock, 2015). must be considered. For instance, the incorporation of small
DNA (~50 bp) elements known as intercistronic expression elements between genes
(Zhou et al., 2007) has been shown to direct the fragmentation of a larger precursor
transcript into smaller transcripts whereby translation is improved of each gene (Lu et al.,
2013; Zhou et al., 2007).
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A number of novel metabolic pathways have now been successfully introduced
into plants through plastid transformation, such as the production of polyhydroxybutyrate
(Lössl et al., 2003, 2005); the enhancement of β-carotene content (Apel and Bock, 2009;
Harada et al., 2014; Wurbs et al., 2007); the introduction of the mevalonate pathway
(Kumar et al., 2012); the biosynthesis of artemisinic acid for the production of
artemisinin (Fuentes et al., 2016; Saxena et al., 2014); the increased production of
vitamin E in tobacco (Lu et al., 2013) and lettuce (Yabuta et al., 2013); the expression of
dhurrin, a cyanogenic glucoside found in Sorghum bicolor (Gnanasekaran et al., 2016);
and the production of squalene, a triterpene (Pasoreck et al., 2016) (see Table 3 for
details).
A significant amount of research is also focusing on increasing crop yields by
improving photosynthesis and reducing photorespiration (Longoni et al., 2015; Ort et al.,
2015; Sharwood et al., 2016). Ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco)—the enzyme that carries out CO2 fixation plus the competing oxygenase
reaction—has a relatively low turnover number for the carboxylation reaction (Carmo-
Silva et al., 2015; Occhialini et al., 2016; Whitney et al., 2015; Wilson et al., 2016).
Lower photosynthetic organisms, such as cyanobacteria, express a catalytically more
active Rubisco that is reliant on an efficient carbon concentrating mechanism (CCM) for
cell survival (Price and Howitt, 2014). Therefore, there is current interest in replacing the
higher plant Rubisco with a cyanobacterial version and its CCM (Price et al., 2013;
Raines, 2011). Recently, Lin et al. (2014) successfully swapped the tobacco plastid rbcL
gene coding for the large subunit of Rubisco with genes from the cyanobacterium
Synechococcus elongatus PCC 7942 coding for the large subunit of Rubisco. Co-
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expression of the Rubisco assembly factor RbcX or the CCM-associated protein CcmM
were found to have little influence on S. elongatus PCC 7942 Rubisco assembly in leaf
chloroplasts (Occhialini et al., 2016). In the absence of a CCM, the resulting plants were
capable of growing photoautotrophically only at elevated levels of CO2 (see Carmo-Silva
et al., 2015 and Ort et al., 2015 for further studies).
Factors limiting the technology
Narrow range of transformable plant species
Routine chloroplast transformation is currently limited to dicotyledonous plants and
mainly members of the Solanaceae family (see Table 2 for details). Although
transformation of several crop plants has been successfully demonstrated, it is not yet
available for monocotyledonous plants except rice (Lee et al., 2006), where it is still
limited to ‘proof of concept’.
The major reasons for the lack of plastid transformation protocols for cereals
appear to be the recalcitrant nature of these species to existing regeneration protocols
(Ahmadabadi et al., 2007; Lee et al., 2006) and their natural resistance to the current
antibiotics used for chloroplast transformation (Fromm et al., 1987; Li et al., 2010). The
recent observation that plastids can move between cells (Bock, 2010; Stegemann and
Bock, 2009) raises the possibility of grafting donor plant tissue containing transformed
plastids on to a recipient plant tissue, thus allowing the migration of transplastomic
plastids into the untransformed stock (Stegemann et al., 2012). Using this approach,
Thyssen et al. (2012) were able to transfer plastids carrying the aadA gene and aurea
young leaf colour phenotype (barau gene) from Nicotiana sylvestris (donor) to Nicotiana
tabacum (recipient). Although incompatible nucleus–plastid combinations may result in
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adverse phenotypic effects in the resulting progeny (Greiner and Bock, 2013; Pelletier
and Budar, 2007), this should not be problematic within closely related species where the
transfer of chloroplasts has been demonstrated to be functional (Bock, 2014).
Poor expression of transgenes in non-green plastids
Another major limitation is that expression of transgenes in non-green plastids is not as
efficient as in green plastids (chloroplasts). Although the plastome is relatively small, its
contribution to protein expression in photosynthetically active tissues can reach as high as
50% of the total leaf protein content. In contrast, expression levels are substantially lower
in non-green plastids (Zhang et al., 2012), which can result in poorer expression of
foreign proteins. For example, expression of the HIV antigen p24 fused with Nef (p24-
Nef) driven from the 16S rRNA promoter, Prrn, was observed to be 2.5% TSP in green
tomatoes but was not detected in ripe fruit (chromoplasts) (Zhou et al., 2008). However,
the aadA selectable marker could be expressed at ~50% the levels found in green plastids
(Ruf et al., 2001).
A genome-wide analysis of tomato fruits and potato tubers has shown that almost
all genes in non-green plastids are strongly down-regulated apart from two essential
genes, accD and clpP, involved in lipid metabolism and the plastid proteolytic
machinery, respectively (Kahlau and Bock, 2008; Valkov et al., 2009), both of which
play important functions in non-green plastids (Ohlrogge and Browse, 1995; Sakamoto,
2006). Therefore, transgene expression using the promoters and 5′-untranslated regions of
these genes may help to overcome some of the challenges related to poor gene expression
in non-green plastids.
Transgene containment in the chloroplast genome is not absolute
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The attraction of using plants as ‘green factories’ lies in their scalability. Although
plastids in principle provide a degree of natural gene containment due to their maternal
mode of inheritance, this containment is not absolute, as mentioned earlier. Despite this,
it is still considered highly unlikely that transgenes from a transplastome will enter into
the germplasm of weedy relatives under field conditions (Daniell, 2007). The barriers
include sexual incompatibility between a crop and its weedy relatives, the prevalence of
selection pressure, and desynchronized flowering patterns. Even after hybridization, a
successful introgression event requires that the F1 hybrid survive to produce at least one
single backcross (BC1) hybrid. The higher the number of backcrosses, the quicker the
introgression of a gene will be. Furthermore, the incoming gene must confer a selective
advantage to the host plant; otherwise, natural rearrangements at the chromosome level
will result in its elimination from the plant genome (see Stewart et al., 2003 for a detailed
review).
In order to address potential concerns over gene containment, strategies are being
developed to deliver speed and scalability as well as absolute gene containment without
recourse to costly greenhouse facilities. One such platform is the use of temporary
immersion bioreactors (TIBs). This novel plant biomass propagation technique provides
an alternative way of obtaining rootless leafy plant biomass within a short timeframe of
just 40 days. Expression of green fluorescent protein (GFP) and TetC in leaves obtained
through TIBs was only 50% lower than that of the leaves of plants grown in pots.
However, when the accumulation of these proteins was normalized in terms of growth
area, the productivity of TIBs was around 80–100-fold higher than the plants grown in
soil (Michoux et al., 2011), suggesting that the TIB-based regeneration system could be a
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potential route of obtaining high levels of recombinant proteins at low cost under
absolutely contained conditions.
Another advantage of the use of TIBs is that plant tissue is grown in the presence
of sucrose as a carbon source, which means that non-autotrophic mutants expressing high
levels of a foreign protein can still be propagated (Michoux et al., 2013). This technology
is now under further refinement and optimization to elucidate the kinetics of cell-to-
plantlet morphogenesis and accumulation of recombinant proteins.
Limited availability of inducible gene expression systems
The high level of recombinant protein expression sometimes observed with chloroplast
transformation can place a metabolic burden on the plant, leading to unintended
phenotypes such as poorer growth (Tregoning et al., 2003) and high light sensitivity
(Ahmad et al., 2012b). This makes tightly controllable expression systems highly
desirable. Although attempts have been made to repress/control the expression of plastid
genes, they have relied on the chemical induction of transgenes introduced into the
nucleus and the subsequent import of their product into plastids. Therefore, this approach
requires transformation of both cellular compartments (Buhot et al., 2006; Gottschamel et
al., 2016; Lössl et al., 2005; McBride et al., 1994; Muhlbauer and Koop, 2005). The first
‘plastid-only’ strategy for an inducible expression system used a modified form of an E.
coli theophylline-binding thiamine pyrophosphate translational riboswitch (Verhounig et
al., 2010). The synthetic riboswitch was able to deliver tight regulatory control over
transgene expression, in this case GFP, albeit with a lower than normal level of GFP
accumulation when translation was induced with theophylline (0.01─0.02% TSP; 10-fold
lower than the standard expression) (Verhounig et al., 2010).
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Absence of glycosylation
Plastids are capable of carrying out many of the post-translational modifications (PTMs)
needed for the production of a physiologically active protein, such as disulphide bond
formation, lipidation, multimerization, and N-terminal methionine excision (Rigano et
al., 2012). However, chloroplasts lack the necessary machinery to carry out
glycosylation, one of the most important PTMs in eukaryotes (Paul and Ma, 2011). The
absence of glycosylation in chloroplasts may therefore represent a bottleneck for the
synthesis of those proteins that are dependent on this PTM for proper functioning.
However, experiments have shown that glycosylation is not always a stringent
requirement for protein function. For example, xylanases are single-chain glycoproteins,
ranging in size from 6–80 kDa, which are active at pH 4.5–6.5 and 40–60 °C.
Transplastomic expression of an alkali-thermostable xylanase from Bacillus subtilis
strain NG-27 in tobacco plants resulted in its accumulation to 6% TSP. The chloroplast-
expressed xylanase was shown to be as active as the native version (Leelavathi et al.,
2003). Another study expressed type I interferon α2b (IFN-α2b)—a member of the
human cytokine glycoprotein family—in tobacco chloroplasts and observed that the
chloroplast-made INF-α2b induced up-regulation of major histocompatibility complex
class I molecules and activation of natural killer cells in a similar fashion to a
commercial-grade glycosylated preparation (Arlen et al., 2007). However, more studies
are needed to determine the effect of glycosylation on protein stability by taking a range
of native glycoproteins and then comparing them with the unglycosylated versions by
expressing them in chloroplasts. Alternatively, in-vitro glycosylation can be performed
on purified chloroplast-made proteins (Strasser et al., 2014).
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Degradation of foreign proteins
Plastids have evolved a complex network of biological pathways capable of maintaining
the dynamic equilibrium of the protein pool, thus allowing the cell to effectively
acclimate to environmental changes (De Marchis et al., 2012). Protein degradation plays
a crucial role in maintaining the functional integrity of the plastid proteome, for example,
through the replacement of photo-damaged photosystem II subunits such as D1 (reviewed
in Nixon et al., 2010) and the removal of unassembled subunits of photosynthetic
complexes to control protein abundance, as well as during plant senescence (Jarvis and
López-Juez, 2013).
The stroma of the chloroplast contains many proteases (Adam and Sakamoto,
2014; Sakamoto, 2006), which have the potential to degrade recombinant proteins
produced in the chloroplast (Bellucci et al., 2005; Birch-Machin et al., 2004; Elghabi et
al., 2011). Studies into the mechanism governing protein stability in plastids have
revealed that the N-terminus of a protein harbours sequences that can influence protein
stability and turnover in plastids (Apel et al., 2010). Addition of five amino acids (Met-
Ala-Ser-Ile-Ser) to the N-terminus of VP6 increased protein accumulation >16% TSP
(Borchers et al., 2012), and fusing GFP and PlyGBS residues to the N- and C- terminal
sequences of cyanovirin-N, which in vivo irreversibly inhibits fusion of HIV particles
with target cells, now permitted its accumulation (Elghabi et al., 2011). Similarly, human
insulin was rapidly lost from chloroplasts unless fused with cholera toxin β subunit
(Ruhlman et al., 2007).
Conclusion and perspectives
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Despite notable achievements at the laboratory scale, chloroplast transformation
technology has not yet reached the field. Construction of chloroplast expression vectors,
their delivery into the chloroplast genome, and recovery of homoplasmic plant lines is a
relatively straightforward but lengthy process. Current attempts to streamline this process
include the adoption of high-throughput cloning methods for the construction of
chloroplast expression vectors (Gottschamel et al., 2013; Vafaee et al., 2014) and finding
new selectable markers (Bellucci et al., 2015; Li et al., 2010). Downstream technologies
are also being developed, such as protein purification from transplastomic plants (Ahmad
et al., 2012a) and growth of plant tissue in bioreactors under absolute transgene
containment (Michoux et al., 2011; 2013). The ability to transform non-green plastids
(Kumar et al., 2004a; Wurbs et al., 2007; Zhang et al., 2012) should allow this
technology to have an impact on diverse metabolic processes in plants (Warzecha, 2016).
The recent demonstrations that intact long double-stranded RNA (dsRNA) can be
produced in plastids (Jin et al., 2015; Zhang et al., 2015) has opened up new vistas to
control insect population through RNA interference (RNAi). Using viral vectors (Saxena
et al., 2016) to transiently express and target recombinant proteins to different cellular
organelles such as mitochondria, chloroplasts, and nucleus simultaneously (Majer et al.,
2015) provides another route of using plants as cellular factories.
There is increasing interest in using photoautotrophs such as cyanobacteria,
microalgae, and plants as expression platforms for synthetic biology (Scharff and Bock,
2014; Sinagawa-García et al., 2009; Verhounig et al., 2010). Chloroplast synthetic
biology is still in its infancy, but progress is being made to develop the necessary tools to
control the expression of foreign genes in the plastome. Ultimately, artificial plastomes
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could be engineered to generate new types of plastid, such as a ‘nitroplast’ specialized in
nitrogen fixation. Replacing the whole genome may not be straightforward due to
homologous recombination between the resident and incoming synthetic plastid genomes,
which will result in a hybrid genome, as observed in whole-genome transplantation in
Chlamydomonas reinhardtii (O’Neill et al., 2012).
One area where chloroplast transformation holds greater immediate promise is the
production of plant-based edible vaccines (Waheed et al., 2015). Although a fairly large
number of vaccine antigens (see Lössl and Waheed, 2011; Scotti et al., 2012; Waheed et
al., 2015 for detailed reviews) have been expressed in higher plant chloroplasts (Table 1),
none of the chloroplast-made proteins has completed clinical trials. Due to the high
standards for human application, it is likely that plastid-made vaccines for veterinary
application may come to market earlier (Clarke et al., 2013). One of the possible reasons
for the delay of plastid-derived vaccines is that the technology is heavily patented. For
example, Chlorogen (St. Louis, MO, USA), now a defunct biotech start-up, acquired or
licensed a vast portfolio of intellectual property relating to chloroplast transformation.
The sale of this company’s assets may allow a new player to attempt to commercialize
this process in the future (Paul and Ma, 2011).
Acknowledgments
NA would like to thank the Higher Education Commission (Pakistan) for financial
support. FM and PN are grateful to the Biotechnology and Biological Sciences Research
Council (UK) for funding their research work. AL is grateful to the Norwegian Research
Council (GLOBVAC Program). The authors sincerely apologize to all those colleagues
whose work could not be discussed here due to space constraints.
Page 20
References
Adam Z, Sakamoto W. 2014. Plastid proteases. In: Theg, SM, Wollman, FA, eds.
Plastid biology. New York: Springer, 359–389.
Ahmad N, Michoux F, McCarthy J, Nixon PJ. 2012a. Expression of the affinity tags,
glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts.
Planta 235, 863–871.
Ahmad N, Michoux F, Nixon PJ. 2012b. Investigating the production of foreign
membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal
oxidase. PLoS One 7, e41722.
Ahmadabadi M, Ruf S, Bock R. 2007. A leaf-based regeneration and transformation
system for maize (Zea mays L.). Transgenic Research 16, 437–448.
Albarracín RM, Becher ML, Farran I, Sander VA, Corigliano MG, Yácono ML,
Pariani S, López ES, Veramendi J, Clemente M. 2015. The fusion of
Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock
protein 83-kDa improves expression levels in tobacco chloroplasts.
Biotechnology Journal 10, 748–759.
Allainguillaume J, Harwood T, Ford CS, Cuccato G, Norris C, Allender CJ, Welters
R, King GJ, Wilkinson MJ. 2009. Rapeseed cytoplasm gives advantage in wild
relatives and complicates genetically modified crop biocontainment. New
Phytologist 183, 1201–1211.
Apel W, Bock R. 2009. Enhancement of carotenoid biosynthesis in transplastomic
tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiology 151,
59–66.
Page 21
Apel W, Schulze WX, Bock R. 2010. Identification of protein stability determinants in
chloroplasts. The Plant Journal 63, 636–650.
Arakawa T, Chong DKX, Lawrence Merritt J, Langridge WHR. 1997. Expression of
cholera toxin B subunit oligomers in transgenic potato plants. Transgenic
Research 6, 403–413.
Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H.
2007. Field production and functional evaluation of chloroplast-derived
interferon-α2b. Plant Biotechnology Journal 5, 511–525.
Arnold W, Rump A, Klipp W, Priefer UB, Puhler A. 1988. Nucleotide sequence of a
24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster
of Klebsiella pneumoniae. Journal of Molecular Biology 203, 715–738.
Azhagiri AK, Maliga P. 2007. Exceptional paternal inheritance of plastids in
Arabidopsis suggests that low-frequency leakage of plastids via pollen may be
universal in plants. The Plant Journal 52, 817–823.
Bellucci M, De Marchis F, Ferradini N, Pompa A, Veronesi F, Rosellini D. 2015. A
mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase
confers gabaculine resistance when expressed in tobacco plastids. Plant Cell
Reports 34, 2127–2136.
Bellucci M, De Marchis F, Mannucci R, Bock R, Arcioni S. 2005. Cytoplasm and
chloroplasts are not suitable subcellular locations for beta-zein accumulation in
transgenic plants. Journal of Experimental Botany 56, 1205–1212.
Page 22
Birch-Machin I, Newell CA, Hibberd JM, Gray JC. 2004. Accumulation of rotavirus
VP6 protein in chloroplasts of transplastomic tobacco is limited by protein
stability. Plant Biotechnology Journal 3, 261–270.
Bock R. 2010. The give-and-take of DNA: horizontal gene transfer in plants. Trends in
Plant Science 15, 11–22.
Bock R. 2013. Strategies for metabolic pathway engineering with multiple transgenes.
Plant Molecular Biology 83, 1–11.
Bock R. 2014. Genetic engineering of the chloroplast: novel tools and new applications.
Current Opinion in Biotechnology 26, 7–13.
Bock R. 2015. Engineering Plastid Genomes: Methods, tools, and applications in basic
research and biotechnology. Annual Review of Plant Biology 66, 211–241.
Bohmert-Tatarev K, McAvoy S, Daughtry S, Peoples OP, Snell KD. 2011. High
levels of bioplastic are produced in fertile transplastomic tobacco plants
engineered with a synthetic operon for the production of polyhydroxybutyrate.
Plant Physiology 155, 1690–1708.
Borchers AMI, Gonzalez‐Rabade N, Gray JC. 2012. Increased accumulation and
stability of rotavirus VP6 protein in tobacco chloroplasts following changes to the
5′ untranslated region and the 5′ end of the coding region. Plant Biotechnology
Journal 10, 422–434.
Boynton JE, Gillham NW, Harris EH, et al. 1988. Chloroplast transformation in
Chlamydomonas with high velocity microprojectiles. Science 240, 1534–1538.
Buhot L, Horvath E, Medgyesy P, Lerbs-Mache S. 2006. Hybrid transcription system
for controlled plastid transgene expression. The Plant Journal 46, 700 –707.
Page 23
Carbonell-Caballero J, Alonso R, Ibañez V, Terol J, Talon M, Dopazo J. 2015. A
phylogenetic analysis of 34 chloroplast genomes elucidates the relationships
between wild and domestic species within the genus citrus. Molecular Biology
and Evolution 32, 2015–2035.
Carmo-Silva E, Scales JC, Madgwick PJ, Parry MAJ. 2015. Optimizing Rubisco and
its regulation for greater resource use efficiency. Plant Cell and Environment 38,
1817–1832.
Castiglia D, Sannino L, Marcolongo L, Ionata E, Tamburino R, Stradis A, Cobucci-
Ponzano B, Moracci M, Cara F, Scotti N. 2016. High-level expression of
thermostable cellulolytic enzymes in tobacco transplastomic plants and their use
in hydrolysis of an industrially pretreated Arundo donax L. biomass.
Biotechnology for Biofuels 9, 154–170.
Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z, Maliga P. 2006. Expression of
the cry9Aa2 Bt gene in tobacco chloroplasts confers resistance to potato tuber
moth. Transgenic Research 15, 481–488.
Chan H-T, Xiao Y, Weldon WC, Oberste SM, Chumakov K, Daniell H. 2016. Cold
chain and virus free chloroplast-made booster vaccine to confer immunity against
different polio virus serotypes. Plant Biotechnology Journal doi:
10.1111/pbi.12575
Chen PJ, Senthilkumar R, Jane WN, He Y, Tian Z, Yeh KW. 2014. Transplastomic
Nicotiana benthamiana plants expressing multiple defence genes encoding
protease inhibitors and chitinase display broad-spectrum resistance against
insects, pathogens and abiotic stresses. Plant Biotechnology Journal 12, 503–515.
Page 24
Cieslewicz MJ, Kasper DL, Wang Y, Wessels MR. 2001. Functional analysis in type Ia
group B streptococcus of a cluster of genes involved in extracellular
polysaccharide production by diverse species of Streptococci. Journal of
Biological Chemistry 276, 139–146.
Clarke JL, Waheed MT, Lössl AG, Martinussen I, Daniell H. 2013. How can plant
genetic engineering contribute to cost-effective fish vaccine development for
promoting sustainable aquaculture? Plant Molecular Biology 83, 33–40.
Daniell H. 2007. Transgene containment by maternal inheritance: effective or elusive?
Proceedings of the National Academy of Sciences of the United States of America
104, 6879–6880.
Daniell H, Datta R, Varma S, Gray S, Lee SB. 1998. Containment of herbicide
resistance through genetic engineering of the chloroplast genome. Nature
Biotechnology 16, 345–348.
Daniell H, Lee SB, Panchal T, Wiebe PO. 2001a. Expression of the native cholera toxin
B subunit gene and assembly as functional oligomers in transgenic tobacco
chloroplasts. Journal of Molecular Biology 311, 1001–1009.
Daniell H, Muthukumar B, Lee SB. 2001b. Marker free transgenic plants: engineering
the chloroplast genome without the use of antibiotic selection. Current Genetics
39, 109–116.
Day A, Goldschmidt‐Clermont M. 2011. The chloroplast transformation toolbox:
selectable markers and marker removal. Plant Biotechnology Journal 9, 540–553.
Page 25
De Cosa B, Moar W, Lee SB, Miller M, Daniell H. 2001. Overexpression of the Bt
cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nature
Biotechnology 19, 71–74.
De Marchis F, Pompa A, Bellucci M. 2012. Plastid proteostasis and heterologous
protein accumulation in transplastomic plants. Plant Physiology 160, 571–581.
De Marchis F, Wang Y, Stevanato P, Arcioni S, Bellucci M. 2008. Genetic
transformation of the sugar beet plastome. Transgenic Research 18, 17–30.
DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H. 2001. Expression of an
antimicrobial peptide via the chloroplast genome to control phytopathogenic
bacteria and fungi. Plant Physiology 127, 852–862.
Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo JM, Tissot G. 2004.
Generation of fertile transplastomic soybean. Plant Molecular Biology 55, 479–
489.
Dufourmantel N, Tissot G, Goutorbe F, Garcon F, Muhr C, Jansens S, Pelissier B,
Peltier G, Dubald M. 2005. Generation and analysis of soybean plastid
transformants expressing Bacillus thuringiensis Cry1Ab protoxin. Plant
Molecular Biology 58, 659–668.
Elghabi Z, Karcher D, Zhou F, Ruf S, Bock R. 2011. Optimization of the expression of
the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome. Plant
Biotechnology Journal 9, 599–608.
Espinoza-Sánchez EA, Álvarez-Hernández MH, Torres-Castillo JA, Rascón-Cruz
Q, Gutiérrez-Díez A, Zavala-García F, Sinagawa-García SR. 2015. Stable
Page 26
expression and characterization of a fungal pectinase and bacterial peroxidase
genes in tobacco chloroplast. Electronic Journal of Biotechnology 18, 161–168.
Espinoza-Sánchez EA, Torres-Castillo JA, Rascón-Cruz Q, Zavala-García F,
Sinagawa-García SR. 2016. Production and characterization of fungal β-
glucosidase and bacterial cellulases by tobacco chloroplast transformation. Plant
Biotechnology Reports 10, 61–73.
Farran I, McCarthy SI, Rìo-Manterola F, Mansilla C, Lasarte JJ, Mingo-Castel M.
2010. The vaccine adjuvant extra domain A from fibronectin retains its
proinflammatory properties when expressed in tobacco chloroplasts. Planta 231,
977–990.
Farran I, Río-Manterola F, Íñiguez M, Gárate S, Prieto J, Mingo-Castel AM. 2008.
High-density seedling expression system for the production of bioactive human
cardiotrophin-1, a potential therapeutic cytokine, in transgenic tobacco
chloroplasts. Plant Biotechnology Journal 6, 516–527.
Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H. 2003. A chloroplast
transgenic approach to hyper-express and purify Human Serum Albumin, a
protein highly susceptible to proteolytic degradation. Plant Biotechnology Journal
1, 71–79.
Fromm H, Edelman M, Aviv D, Galun E. 1987. The molecular basis for rRNA-
dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO Journal 6,
3233–3237.
Page 27
Fuentes P, Zhou F, Erban A, Karcher D, Kopka J, Bock R. 2016. A new synthetic
biology approach allows transfer of an entire metabolic pathway from a medicinal
plant to a biomass crop. eLife 5, e13664.
Garton S, Knight H, Warren GJ, Knight MR, Thorlby GJ. 2007. crinkled leaves 8 –
A mutation in the large subunit of ribonucleotide reductase – leads to defects in
leaf development and chloroplast division in Arabidopsis thaliana. The Plant
Journal 50, 118–127.
Gilbert N. 2013. A hard look at GM crops. Nature 497, 24–26.
Gisby MF, Mudd EA, Day A. 2012. Growth of transplastomic cells expressing D-amino
acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine.
Plant Physiology 160, 2219–2226.
Gnanasekaran T, Karcher D, Nielsen AZ, Martens HJ, Ruf S, Kroop X, Olsen CE,
Motawie MS, Pribil M, Møller BL. 2016. Transfer of the cytochrome P450-
dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum
chloroplasts for light-driven synthesis. Journal of Experimental Botany 67, 2495–
2506.
Gorantala J, Grover S, Rahi A, Chaudhary P, Rajwanshi R, Sarin NB, Bhatnagar
R. 2014. Generation of protective immune response against anthrax by oral
immunization with protective antigen plant-based vaccine. Journal of
Biotechnology 176, 1–10.
Gottschamel J, Lössl A, Ruf S, Wang Y, Skaugen M, Bock R, Clarke JL. 2016.
Production of dengue virus envelope protein domain III-based antigens in tobacco
Page 28
chloroplasts using inducible and constitutive expression systems. Plant Molecular
Biology 91, 497–512.
Gottschamel J, Waheed M, Clarke J, Lössl A. 2013. A novel chloroplast
transformation vector compatible with the Gateway® recombination cloning
technology. Transgenic Research 22, 1273–1278.
Gray BN, Ahner BA, Hanson MR. 2009. High-level bacterial cellulase accumulation in
chloroplast-transformed tobacco mediated by downstream box fusions.
Biotechnology and Bioengineering 102, 1045–1054.
Greiner S, Bock R. 2013. Tuning a ménage à trois: co‐evolution and co‐adaptation of
nuclear and organellar genomes in plants. BioEssays 35, 354–365.
Greiner S, Sobanski J, Bock R. 2015. Why are most organelle genomes transmitted
maternally? BioEssays 37, 80–94.
Guda C, Lee SB, Daniell H. 2000. Stable expression of a biodegradable protein-based
polymer in tobacco chloroplasts. Plant Cell Reports 19, 257–262.
Hagemann R. 2004. The sexual inheritance of plant organelles. In: Daniell H, Chase C,
eds. Molecular biology and biotechnology of plant organelles: chloroplasts and
mitochondria. Dordrecht: Springer, 93–113.
Haq TA, Mason HS, Clements JD, Arntzen CJ. 1995. Oral immunization with a
recombinant bacterial antigen produced in transgenic plants. Science 268, 714–
716.
Harada H, Maoka T, Osawa A, Hattan J, Kanamoto H, Shindo K, Otomatsu T,
Misawa N. 2014. Construction of transplastomic lettuce (Lactuca sativa)
Page 29
dominantly producing astaxanthin fatty acid esters and detailed chemical analysis
of generated carotenoids. Transgenic Research 23, 303–315.
Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM. 2003.
Chloroplast transformation in oilseed rape. Transgenic Research 12, 111–114.
Huang CY, Ayliffe MA, Timmis JN. 2003. Direct measurement of the transfer rate of
chloroplast DNA into the nucleus. Nature 422, 72–76.
Hussein S, Ruiz ON, Terry N, Daniell H. 2007. Phytoremediation of mercury and
organomercurials in chloroplast transgenic plants: enhanced root uptake,
translocation to shoots, and volatilization. Environmental Science and Technology
41, 8439–8446.
Iamtham S, Day A. 2000. Removal of antibiotic resistance genes from transgenic
tobacco plastids. Nature Biotechnology 18, 1172–1176.
Inka Borchers AM, Gonzalez‐Rabade N, Gray JC. 2012. Increased accumulation and
stability of rotavirus VP6 protein in tobacco chloroplasts following changes to the
5′ untranslated region and the 5′ end of the coding region. Plant Biotechnology
Journal 10, 422–434.
Jaffe B, Kovacs K, Andras C, Bodi Z, Liu Z, Fray RG. 2008. Methylation of
chloroplast DNA does not affect viability and maternal inheritance in tobacco and
may provide a strategy towards transgene containment. Plant Cell Reports 27,
1377–1384.
Jarvis P. 2004. Organellar proteomics: chloroplasts in the spotlight. Current Biology 14,
R317–R319.
Page 30
Jarvis P, López-Juez E. 2013. Biogenesis and homeostasis of chloroplasts and other
plastids. Nature Reviews: Molecular Cell Biology 14, 787–802.
Jin S, Daniell H. 2014. Expression of gamma-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. Plant Biotechnology Journal 12, 1274–1285.
Jin S, Kanagaraj A, Verma D, Lange T, Daniell H. 2011. Release of hormones from
conjugates: chloroplast expression of β-glucosidase results in elevated
phytohormone levels associated with significant increase in biomass and
protection from aphids or whiteflies conferred by sucrose esters. Plant Physiology
155, 222–235.
Jin S, Singh ND, Li L, Zhang X, Daniell H. 2015. Engineered chloroplast dsRNA
silences cytochrome p450 monooxygenase, V‐ATPase and chitin synthase genes
in the insect gut and disrupts Helicoverpa armigera larval development and
pupation. Plant Biotechnology Journal 13, 435–446.
Jin S, Zhang X, Daniell H. 2012. Pinellia ternata agglutinin expression in chloroplasts
confers broad spectrum resistance against aphid, whitefly, Lepidopteran insects,
bacterial and viral pathogens. Plant Biotechnology Journal 10, 313–327.
Kahlau S, Bock R. 2008. Plastid transcriptomics and translatomics of tomato fruit
development and chloroplast-to-chromoplast differentiation: chromoplast gene
expression largely serves the production of a single protein. The Plant Cell 20,
856–874.
Page 31
Karimi F, Mousavi A, Salmanian A, Alizadeh H, Rafati S. 2013. Immunogenicity of
EIT chimeric protein expressed in transplastomic tobacco plants towards
development of an oral vaccine against Escherichia coli O157:H7. Plant
Biotechnology Reports 7, 535–546.
Khan M, Hameed W, Nozoe M, Shiina T. 2007. Disruption of the psbA gene by the
copy correction mechanism reveals that the expression of plastid-encoded genes is
regulated by photosynthesis activity. Journal of Plant Research 120, 421–430.
Khan MS, Kanwal B, Nazir S. 2015. Metabolic engineering of the chloroplast genome
reveals that the yeast ArDH gene confers enhanced tolerance to salinity and
drought in plants. Frontiers in Plant Science 6, 725.
Kleffmann T, Russenberger D, von Zychlinski A, Christopher W, Sjölander K,
Gruissem W, Baginsky S. 2004. The Arabidopsis thaliana chloroplast proteome
reveals pathway abundance and novel protein functions. Current Biology 14, 354–
362.
Kohli A, Miro B, Twyman RM. 2010. Transgene integration, expression and stability in
plants: strategies for improvements. In: Kole C, Michler CH, Abbott AG, Hall
TC, eds. Transgenic crop plants. Heidelberg: Springer, 201–237.
Kolotilin I, Kaldis A, Devriendt B, Joensuu J, Cox E, Menassa R. 2012. Production of
a subunit vaccine candidate against porcine post-weaning diarrhea in high-
biomass transplastomic tobacco. PLoS One 7, e42405.
Kota M. 1999. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in
chloroplasts confers resistance to plants against susceptible and Bt-resistant
Page 32
insects. Proceedings of the National Academy of Sciences of the United States of
America 96, 1840–1845.
Krichevsky A, Meyers B, Vainstein A, Maliga P, Citovsky V. 2010. Autoluminescent
plants. PLoS One 5, e15461.
Kumar S, Dhingra A, Daniell H. 2004a. Plastid-expressed betaine aldehyde
dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced
salt tolerance. Plant Physiology 136, 2843–2854.
Kumar S, Dhingra A, Daniell H. 2004b. Stable transformation of the cotton plastid
genome and maternal inheritance of transgenes. Plant Molecular Biology 56, 203–
216.
Kumar S, Hahn FM, Baidoo E, Kahlon TS, Wood DF, McMahan CM, Cornish K,
Keasling JD, Daniell H, Whalen MC. 2012. Remodeling the isoprenoid pathway
in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.
Metabolic Engineering 14, 19–28.
Kwon KC, Nityanandam R, New JS, Daniell H. 2013. Oral delivery of bioencapsulated
exendin‐4 expressed in chloroplasts lowers blood glucose level in mice and
stimulates insulin secretion in beta‐TC6 cells. Plant Biotechnology Journal 11,
77–86.
Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H. 2013. Low cost tuberculosis
vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation,
stability and functional evaluation in vitro. PLoS One 8, e54708.
Le Martret B, Poage M, Shiel K, Nugent GD, Dix PJ. 2011. Tobacco chloroplast
transformants expressing genes encoding dehydroascorbate reductase, glutathione
Page 33
reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism
and improved abiotic stress tolerance. Plant Biotechnology Journal 9, 661–673.
Lee S-B, Li B, Jin S, Daniell H. 2011. Expression and characterization of antimicrobial
peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and
bacterial infections. Plant Biotechnology Journal 9, 100–115.
Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H.
2003. Accumulation of trehalose within transgenic chloroplasts confers drought
tolerance. Molecular Breeding 11, 1–13.
Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim
M. 2006. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza
sativa) and transmission of transgenes to their progeny. Molecules and Cells 21,
401–410.
Leelavathi S, Gupta N, Maiti S, Ghosh A, Reddy VS. 2003. Overproduction of an
alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery
of the enzyme. Molecular Breeding 11, 59–67.
Leelavathi S, Reddy VS. 2003. Chloroplast expression of His-tagged GUS-fusions: a
general strategy to overproduce and purify foreign proteins using transplastomic
plants as bioreactors. Molecular Breeding 11, 49–58.
Lelivelt CLC, McCabe MS, Newell CA, Desnoo CB, Dun KMP, Birch-Machin I,
Gray JC, Mills KHG, Nugent JM. 2005. Stable plastid transformation in lettuce
(Lactuca sativa L.). Plant Molecular Biology 58, 763–774.
Li W, Ruf S, Bock R. 2010. Chloramphenicol acetyltransferase as selectable marker for
plastid transformation. Plant Molecular Biology 76, 443–451.
Page 34
Lim S, Ashida H, Watanabe R, Inai K, Kim Y-S, Mukougawa K, Fukuda H,
Tomizawa K-I, Ushiyama K-i, Asao H. 2011. Production of biologically active
human thioredoxin 1 protein in lettuce chloroplasts. Plant Molecular Biology 76,
335–344.
Lin MT, Occhialini A, Andralojc PJ, Parry MA, Hanson MR. 2014. A faster Rubisco
with potential to increase photosynthesis in crops. Nature 513, 547–550.
Liu CW, Lin CC, Chen JJ, Tseng MJ. 2007. Stable chloroplast transformation in
cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant
Cell Reports 26, 1733–1744.
Liu CW, Lin CC, Yiu JC, Chen JJ, Tseng MJ. 2008. Expression of a Bacillus
thuringiensis toxin (Cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata
L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella.
Theoretical and Applied Genetics 117, 75–88.
Longoni P, Leelavathi S, Doria E, Reddy VS, Cella R. 2015. Production by tobacco
transplastomic plants of recombinant fungal and bacterial cell-wall degrading
enzymes to be used for cellulosic biomass saccharification. BioMed Research
International doi:10.1155/2015/289759
Lössl A, Bohmert K, Harloff H, Eibl C, Mühlbauer S, Koop HU. 2005. Inducible
trans-activation of plastid transgenes: expression of the R. eutropha phb operon in
transplastomic tobacco. Plant Cell Physiology 46, 1462–1471.
Lössl A, Eibl C, Harloff HJ, Jung C, Koop HU. 2003. Polyester synthesis in
transplastomic tobacco (Nicotiana tabacum L.): significant contents of
Page 35
polyhydroxybutyrate are associated with growth reduction. Plant Cell Reports 21,
891–899.
Lössl AG, Waheed MT. 2011. Chloroplast-derived vaccines against human diseases:
achievements, challenges and scopes. Plant Biotechnology Journal 9, 527–539.
Lu Y, Rijzaani H, Karcher D, Ruf S, Bock R. 2013. Efficient metabolic pathway
engineering in transgenic tobacco and tomato plastids with synthetic multigene
operons. Proceedings of the National Academy of Sciences of the United States of
America 110, 623–632.
Madanala R, Gupta V, Pandey AK, Srivastava S, Pandey V, Singh PK, Tuli R. 2015.
Tobacco chloroplasts as bioreactors for the production of recombinant superoxide
dismutase in plants, an industrially useful enzyme. Plant Molecular Biology
Reports 33, 1107–1115.
Majer E, Navarro JA, Daros JA. 2015. A potyvirus vector efficiently targets
recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when
expressed at the amino terminus of the polyprotein. Biotechnology Journal 10,
1792–1802.
Maldaner FR, Aragão FJL, dos Santos FB, Franco OL, Lima MDRQ, de Oliveira
Resende R, Vasques RM, Nagata T. 2013. Dengue virus tetra-epitope peptide
expressed in lettuce chloroplasts for potential use in dengue diagnosis. Applied
Microbiology and Biotechnology 97, 5721–5729.
Maliga P. 2002. Engineering the plastid genome of higher plants. Current Opinion in
Plant Biology 5, 164–172.
Page 36
Maliga P. 2003. Progress towards commercialization of plastid transformation
technology. Trends in Biotechnology 21, 20–28.
Maliga P, Bock R. 2011. Plastid biotechnology: food, fuel, and medicine for the 21st
century. Plant Physiology 155, 1501–1510.
Maliga P, Tungsuchat-Huang T. 2014. Plastid transformation in Nicotiana tabacum
and Nicotiana sylvestris by biolistic DNA delivery to leaves. In: Maliga P, ed.
Chloroplast biotechnology: methods and protocols. New York: Springer, 147–
163.
Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B,
Hasegawa M, Penny D. 2002. Evolutionary analysis of Arabidopsis,
cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands
of cyanobacterial genes in the nucleus. Proceedings of the National Academy of
Sciences of the United States of America 99, 12246–12251.
McBride KE, Schaaf DJ, Daley M, Stalker DM. 1994. Controlled expression of plastid
transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7
RNA polymerase. Proceedings of the National Academy of Sciences of the
United States of America 91, 7301–7305.
McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P. 1995.
Amplification of a chimeric Bacillus gene in chloroplasts leads to an
extraordinary level of an insecticidal protein in tobacco. Nature Biotechnology 13,
362–365.
Michoux F, Ahmad N, Hennig A, Nixon P, Warzecha H. 2013. Production of leafy
biomass using temporary immersion bioreactors: an alternative platform to
Page 37
express proteins in transplastomic plants with drastic phenotypes. Planta 237,
903–908.
Michoux F, Ahmad N, McCarthy J, Nixon PJ. 2011. Contained and high-level
production of recombinant proteins in plant chloroplasts using a temporary
immersion bioreactor. Plant Biotechnology Journal 9, 575–584.
Morgenfeld M, Lentz E, Segretin ME, Alfano EF, Bravo-Almonacid F. 2014.
Translational fusion and redirection to thylakoid lumen as strategies to enhance
accumulation of human papillomavirus E7 antigen in tobacco chloroplasts.
Molecular Biotechnology 56, 1021–1031.
Muhlbauer SK, Koop HU. 2005. External control of transgene expression in tobacco
plastids using the bacterial lac repressor. The Plant Journal 43, 941–946.
Nadai M, Bally J, Vitel M, Job C, Tissot G, Botterman J, Dubald M. 2009. High-
level expression of active human alpha1-antitrypsin in transgenic tobacco
chloroplasts. Transgenic Research 18, 173–183.
Nixon PJ, Michoux F, Yu J, Boehm M, Komenda J. 2010. Recent advances in
understanding the assembly and repair of photosystem II. Annals of Botany 106,
1–16.
Nugent GD, Coyne S, Nguyen TT, Kavanagh TA, Dix PJ. 2006. Nuclear and plastid
transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-
mediated uptake of DNA into protoplasts. Plant Science 170, 135–142.
O’Neill BM, Mikkelson KL, Gutierrez NM, Cunningham JL, Wolff KL, Szyjka SJ,
Yohn CB, Redding KE, Mendez MJ. 2012. An exogenous chloroplast genome
Page 38
for complex sequence manipulation in algae. Nucleic Acids Research 40, 2782–
2792.
Occhialini A, Lin MT, Andralojc PJ, Hanson MR, Parry MAJ. 2016. Transgenic
tobacco plants with improved cyanobacterial Rubisco expression but no extra
assembly factors grow at near wild-type rates if provided with elevated CO2. The
Plant Journal 85, 148–160.
Oey M, Lohse M, Kreikemeyer B, Bock R. 2009a. Exhaustion of the chloroplast
protein synthesis capacity by massive expression of a highly stable protein
antibiotic. The Plant Journal 57, 436–445.
Oey M, Lohse M, Scharff LB, Kreikemeyer B, Bock R. 2009b. Plastid production of
protein antibiotics against pneumonia via a new strategy for high-level expression
of antimicrobial proteins. Proceedings of the National Academy of Sciences of the
United States of America 106, 6579–6584.
Ohlrogge J, Browse J. 1995. Lipid biosynthesis. The Plant Cell 7, 957–970.
Okumura S, Sawada M, Park YW, Hayashi T, Shimamura M, Takase H, Tomizawa
K. 2006. Transformation of poplar (Populus alba) plastids and expression of
foreign proteins in tree chloroplasts. Transgenic Research 15, 637–646.
Ort DR, Merchant SS, Alric J, et al. 2015. Redesigning photosynthesis to sustainably
meet global food and bioenergy demand. Proceedings of the National Academy of
Sciences of the United States of America 112, 8529–8536.
Pasoreck EK, Su J, Silverman IM, Gosai SJ, Gregory BD, Yuan JS, Daniell H. 2016.
Terpene metabolic engineering via nuclear or chloroplast genomes profoundly
Page 39
and globally impacts off‐target pathways through metabolite signalling. Plant
Biotechnology Journal 14, 1862–1875.
Paul M, Ma JK-C. 2011. Plant-made pharmaceuticals: leading products and production
platforms. Biotechnology and Applied Biochemistry 58, 58–67.
Pelletier G, Budar F. 2007. The molecular biology of cytoplasmically inherited male
sterility and prospects for its engineering. Current Opinion in Biotechnology 18,
121–125.
Petersen K, Bock R. 2011. High-level expression of a suite of thermostable cell wall-
degrading enzymes from the chloroplast genome. Plant Molecular Biology 76,
311–321.
Price GD, Howitt SM. 2014. Plant science: Towards turbocharged photosynthesis.
Nature 513, 497–498.
Price GD, Pengelly JJ, Forster B, Du J, Whitney SM, von Caemmerer S, Badger
MR, Howitt SM, Evans JR. 2013. The cyanobacterial CCM as a source of genes
for improving photosynthetic CO2 fixation in crop species. Journal of
Experimental Botany 64, 753–768.
Pyke K. 2009. Plastid biology. Cambridge: Cambridge University Press.
Qin H, Dong Y, von Arnim AG. 2003. Epigenetic interactions between Arabidopsis
transgenes: characterization in light of transgene integration sites. Plant Molecular
Biology 52, 217–231.
Raines CA. 2011. Increasing photosynthetic carbon assimilation in C3 plants to improve
crop yield: current and future strategies. Plant Physiology 155, 36–42.
Page 40
Rey P, Sanz-Barrio R, Innocenti G, Ksas B, Courteille A, Rumeau D, Issakidis-
Bourguet E, Farran I. 2013. Overexpression of plastidial thioredoxins f and m
differentially alters photosynthetic activity and response to oxidative stress in
tobacco plants. Frontiers in Plant Science 4, 390.
Rigano MM, Scotti N, Cardi T. 2012. Unsolved problems in plastid transformation.
Bioengineered 3, 329–333.
Roh KH, Shin KS, Lee YH, Seo SC, Park HG, Daniell H, Lee SB. 2006.
Accumulation of sweet protein monellin is regulated by the psbA 5’ UTRs in
tobacco chloroplasts. Plant Biology 49, 34–43.
Rosales-Mendoza S, Rubio-Infante N, Monreal-Escalante E, Govea-Alonso DO,
García-Hernández AL, Salazar-González JA, González-Ortega O, Paz-
Maldonado LT, Moreno-Fierros L. 2014. Chloroplast expression of an HIV
envelop-derived multiepitope protein: towards a multivalent plant-based vaccine.
Plant Cell Tissue and Organ Culture 116, 111–123.
Rubio-Infante N, Govea-Alonso DO, Alpuche-Solís ÁG, García-Hernández AL,
Soria-Guerra RE, Paz-Maldonado LT, Ilhuicatzi-Alvarado D, Varona-
Santos JT, Verdín-Terán L, Korban SS. 2012. A chloroplast-derived C4V3
polypeptide from the human immunodeficiency virus (HIV) is orally
immunogenic in mice. Plant Molecular Biology 78, 337–349.
Ruf S, Hermann M, Berger IJ, Carrer H, Bock R. 2001. Stable genetic transformation
of tomato plastids and expression of a foreign protein in fruit. Nature
Biotechnology 19, 870–875.
Page 41
Ruf S, Karcher D, Bock R. 2007. Determining the transgene containment level provided
by chloroplast transformation. Proceedings of the National Academy of Sciences,
of the United States of America 104, 6998–7002.
Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H. 2007. Expression of
cholera toxin β-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral
administration protects against development of insulitis in non-obese diabetic
mice. Plant Biotechnology Journal 5, 495–510.
Ruhlman TA, Rajasekaran K, Cary JW. 2014. Expression of chloroperoxidase from
Pseudomonas pyrrocinia in tobacco plastids for fungal resistance. Plant Science
228, 98–106.
Ruiz O. 2002. Optimization of codon composition and regulatory elements for
expression of the human IGF-1 in transgenic chloroplasts. MS Thesis, University
of Florida.
Ruiz ON, Alvarez D, Torres C, Roman L, Daniell H. 2011. Metallothionein expression
in chloroplasts enhances mercury accumulation and phytoremediation capability.
Plant Biotechnology Journal 9, 609–617.
Ruiz ON, Daniell H. 2005. Engineering cytoplasmic male sterility via the chloroplast
genome by expression of β-ketothiolase. Plant Physiology 138, 1232–1246.
Ruiz ON, Hussein HS, Terry N, Daniell H. 2003. Phytoremediation of organomercurial
compounds via chloroplast genetic engineering. Plant Physiology 132, 1344–
1352.
Sakamoto W. 2006. Protein degradation machineries in plastids. Annual Reviews of
Plant Biology 57, 599–621.
Page 42
Saxena B, Subramaniyan M, Malhotra K, Bhavesh NS, Potlakayala SD, Kumar S.
2014. Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and
impact on plant growth. Journal of Biosciences 39, 33–41.
Saxena P, Thuenemann EC, Sainsbury F, Lomonossoff GP. 2016. Virus-derived
vectors for the expression of multiple proteins in plants. In: MacDonald J,
Kolotilin I, Menassa R, eds. Recombinant proteins from plants. New York:
Humana Press, 39–44.
Scharff LB, Bock R. 2014. Synthetic biology in plastids. The Plant Journal 78, 783–798.
Scheid OM, Paszkowski J, Potrykus I. 1991. Reversible inactivation of a transgene in
Arabidopsis thaliana. Molecular Genomics and Genetics 228, 104–112.
Scotti N, Cardi T. 2014. Transgene-induced pleiotropic effects in transplastomic plants.
Biotechnology Letters 36, 229–239.
Scotti N, Rigano MM, Cardi T. 2012. Production of foreign proteins using plastid
transformation. Biotechnology Advances 30, 387–397.
Sharifi Tabar M, Habashi AA, Rajabi Memari H. 2013. Human granulocyte colony-
stimulating factor (hG-CSF) expression in plastids of Lactuca sativa. Iranian
Biomedical Journal 17, 158–164.
Sharwood RE, Ghannoum O, Whitney SM. 2016. Prospects for improving CO2
fixation in C3-crops through understanding C4-Rubisco biogenesis and catalytic
diversity. Current Opinion in Plant Biology 31, 135–142.
Shaver JM, Oldenburg DJ, Bendich AJ. 2006. Changes in chloroplast DNA during
development in tobacco, Medicago truncatula, pea, and maize. Planta 224, 72–82.
Page 43
Sheppard AE, Ayliffe MA, Blatch L, Day A, Delaney SK, Khairul-Fahmy N, Li Y,
Madesis P, Pryor AJ, Timmis JN. 2008. Transfer of plastid DNA to the nucleus
is elevated during male gametogenesis in tobacco. Plant Physiology 148, 328–
336.
Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, Nehra NS. 1999.
Technical advance: stable chloroplast transformation in potato: use of green
fluorescent protein as a plastid marker. The Plant Journal 19, 209–216.
Sikdar S, Serino G, Chaudhuri S, Maliga P. 1998. Plastid transformation in
Arabidopsis thaliana. Plant Cell Reports 18, 20–24.
Sinagawa-García SR, Tungsuchat-Huang T, Paredes-López O, Maliga P. 2009. Next
generation synthetic vectors for transformation of the plastid genome of higher
plants. Plant Molecular Biology 70, 487–498.
Singh A, Verma S, Bansal K. 2010. Plastid transformation in eggplant (Solanum
melongena L.). Transgenic Research 19, 113–119.
Staub JM, Garcia B, Graves J, et al. 2000. High-yield production of a human
therapeutic protein in tobacco chloroplasts. Nature Biotechnology 18, 333–338.
Stegemann S, Bock R. 2009. Exchange of genetic material between cells in plant tissue
grafts. Science 324, 649–651.
Stegemann S, Keuthe M, Greiner S, Bock R. 2012. Horizontal transfer of chloroplast
genomes between plant species. Proceedings of the National Academy of
Sciences of the United States of America 109, 2434–2438.
Steward G. 2000. A new breed of superweed. The Globe and Mail. Toronto: The
Woodbridge Company, retrieved on 2 March 2016
Page 44
Stewart CN, Halfhill MD, Warwick SI. 2003. Transgene introgression from genetically
modified crops to their wild relatives. Nature Reviews Genetics 4, 806–817.
Strasser R, Altmann F, Steinkellner H. 2014. Controlled glycosylation of plant-
produced recombinant proteins. Current Opinion in Biotechnology 30, 95–100.
Su J, Sherman A, Doerfler PA, Byrne BJ, Herzog RW, Daniell H. 2015. Oral delivery
of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses
antibody formation in treatment of Pompe mice. Plant Biotechnology Journal 13,
1023–1032.
Svab Z, Hajdukiewicz P, Maliga P. 1990a. Stable transformation of plastids in higher
plants. Proceedings of the National Academy of Sciences of the United States of
America 87, 8526–8530.
Svab Z, Harper EC, Jones JD, Maliga P. 1990b. Aminoglycoside-3ʹʹ-adenyltransferase
confers resistance to spectinomycin and streptomycin in Nicotiana tabacum. Plant
Molecular Biology 14, 197–205.
Svab Z, Maliga P. 1993. High-frequency plastid transformation in tobacco by selection
for a chimeric aadA gene. Proceedings of the National Academy of Sciences of
the United States of America 90, 913–917.
Svab Z, Maliga P. 2007. Exceptional transmission of plastids and mitochondria from the
transplastomic pollen parent and its impact on transgene containment.
Proceedings of the National Academy of Sciences, of the United States of
America 104, 7003–7008.
Page 45
Thyssen G, Svab Z, Maliga P. 2012. Cell-to-cell movement of plastids in plants.
Proceedings of the National Academy of Sciences of the United States of America
109, 2439–2443.
Tissot G, Canard H, Nadai M, Martone A, Botterman J, Dubald M. 2008.
Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid
lumen of genetically engineered tobacco chloroplasts. Plant Biotechnology
Journal 6, 309–320.
Tregoning JS, Nixon P, Kuroda H, et al. 2003. Expression of tetanus toxin fragment C
in tobacco chloroplasts. Nucleic Acids Research 31, 1174 –1179.
Vafaee Y, Staniek A, Mancheno-Solano M, Warzecha H. 2014. A modular cloning
toolbox for the generation of chloroplast transformation vectors. PLoS One 9,
e110222.
Valkov VT, Scotti N, Kahlau S, MacLean D, Grillo S, Gray JC, Bock R, Cardi T.
2009. Genome-wide analysis of plastid gene expression in potato leaf chloroplasts
and tuber amyloplasts: transcriptional and posttranscriptional control. Plant
Physiology 150, 2030–2044.
Van Dingenen J, De Milde L, Vermeersch M, Maleux K, De Rycke R, De Bruyne M,
Storme V, Gonzalez N, Dhondt S, Inzé D. 2016. Chloroplasts are central players
in sugar-induced leaf growth. Plant Physiology 171, 590–605.
Verhounig A, Karcher D, Bock R. 2010. Inducible gene expression from the plastid
genome by a synthetic riboswitch. Proceedings of the National Academy of
Sciences of the United States of America 107, 6204–6209.
Page 46
Verma D, Kanagaraj A, Jin S, Singh ND, Kolattukudy PE, Daniell H. 2010a.
Chloroplast‐derived enzyme cocktails hydrolyse lignocellulosic biomass and
release fermentable sugars. Plant Biotechnology Journal 8, 332–350.
Verma D, Moghimi B, LoDuca PA, Singh HD, Hoffman BE, Herzog RW, Daniell H.
2010b. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor
formation and fatal anaphylaxis in hemophilia B mice. Proceedings of the
National Academy of Sciences of the United States of America 107, 7101–7106.
Vieler A, Wu G, Tsai C-H, Bullard B, Cornish AJ, Harvey C, Reca I-B, Thornburg
C, Achawanantakun R, Buehl CJ. 2012. Genome, functional gene annotation,
and nuclear transformation of the heterokont oleaginous alga Nannochloropsis
oceanica CCMP1779. PLoS Genetics 8, e1003064.
Viitanen PV. 2004. Metabolic engineering of the chloroplast genome using the
Escherichia coli ubiC gene reveals that chorismate is a readily abundant plant
precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiology 136, 4048–
4060.
Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. 2015. Plastids: the green
frontiers for vaccine production. Frontiers in Plant Science 6, 1005.
Wang D, Lloyd AH, Timmis JN. 2012. Environmental stress increases the entry of
cytoplasmic organellar DNA into the nucleus in plants. Proceedings of the
National Academy of Sciences of the United States of America 109, 2444–2448.
Wang YP, Wei ZY, Zhang YY, Lin CJ, Zhong XF, Wang YL, Ma JY, Ma J, Xing
SC. 2015. Chloroplast-expressed MSI-99 in tobacco improves disease resistance
Page 47
and displays inhibitory effect against rice blast fungus. International Journal of
Molecular Sciences 16, 4628–4641.
Wang YP, Wei ZY, Zhong XF, Lin CJ, Cai YH, Ma J, Zhang YY, Liu YZ, Xing SC.
2016. Stable expression of basic fibroblast growth factor in chloroplasts of
tobacco. International Journal of Molecular Sciences 17, 9–18.
Warzecha H. 2016. Lights, P450, action! Metabolite formation in chloroplasts. Journal
of Experimental Botany 67, 2123–2125.
Waters M, Pyke K. 2005. Plastid development and differentiation. In: Moller SG, ed.
Annual reviews: Plastids. Oxford: Blackwell, 30–59.
Wei Z, Liu Y, Lin C, Wang Y, Cai QA, Dong Y, Xing S. 2011. Transformation of
alfalfa chloroplasts and expression of green fluorescent protein in a forage crop.
Biotechnology Letters 33, 2487–2494.
Whitney SM, Birch R, Kelso C, Beck JL, Kapralov MV. 2015. Improving
recombinant Rubisco biogenesis, plant photosynthesis and growth by
coexpressing its ancillary RAF1 chaperone. Proceedings of the National Academy
of Sciences of the United States of America 112, 3564–3569.
Wicke S, Schneeweiss GM, Müller KF, Quandt D. 2011. The evolution of the plastid
chromosome in land plants: gene content, gene order, gene function. Plant
Molecular Biology 76, 273–297.
Wilson RH, Alonso H, Whitney SM. 2016. Evolving Methanococcoides burtonii
archaeal Rubisco for improved photosynthesis and plant growth. Scientific
Reports doi: 10.1038/srep22284
Page 48
Wurbs D, Ruf S, Bock R. 2007. Contained metabolic engineering in tomatoes by
expression of carotenoid biosynthesis genes from the plastid genome. The Plant
Journal 49, 276–288.
Yabuta Y, Tanaka H, Yoshimura S, Suzuki A, Tamoi M, Maruta T, Shigeoka S.
2013. Improvement of vitamin E quality and quantity in tobacco and lettuce by
chloroplast genetic engineering. Transgenic Research 22, 391–402.
Yácono M, Farran I, Becher ML, Sander V, Sanchez VR, Martín V, Veramendi J,
Clemente M. 2012. A chloroplast-derived Toxoplasma gondii GRA4 antigen
used as an oral vaccine protects against toxoplasmosis in mice. Plant
Biotechnology Journal 10, 1136–1144.
Yarbakht M, Jalali‐Javaran M, Nikkhah M, Mohebodini M. 2015. Dicistronic
expression of human proinsulin–protein A fusion in tobacco chloroplast.
Biotechnology and Applied Biochemistry 62, 55–63.
Yu LX, Gray BN, Rutzke CJ, Walker LP, Wilson DB, Hanson MR. 2007. Expression
of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco.
Journal of Biotechnology 131, 362–369.
Zahin M, Joh J, Khanal S, Husk A, Mason H, Warzecha H, Ghim SJ, Miller DM,
Matoba N, Jenson AB. 2016. Scalable production of HPV16 L1 protein and
VLPs from tobacco leaves. PLoS One 11, e0160995.
Zhang J, Khan SA, Heckel DG, Bock R. 2015. Full crop protection from an insect pest
by expression of long double-stranded RNAs in plastids. Science 347, 991–994.
Page 49
Zhang J, Ruf S, Hasse C, Childs L, Scharff LB, Bock R. 2012. Identification of cis-
elements conferring high levels of gene expression in non-green plastids. The
Plant Journal 72, 115–128.
Zhang XH, Brotherton JE, Widholm JM, Portis AR. 2001. Targeting a nuclear
anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in
enhanced tryptophan biosynthesis: return of a gene to its pre-endosymbiotic
origin. Plant Physiology 127, 131–141.
Zhou F, Badillo-Corona JA, Karcher D, Gonzalez-Rabade N, Piepenburg K,
Borchers AM, Maloney AP, Kavanagh TA, Gray JC, Bock R. 2008. High-
level expression of human immunodeficiency virus antigens from the tobacco and
tomato plastid genomes. Plant Biotechnology Journal 6, 897–913.
Zhou F, Karcher D, Bock R. 2007. Identification of a plastid intercistronic expression
element (IEE) facilitating the expression of stable translatable monocistronic
mRNAs from operons. The Plant Journal 52, 961–972.
Zubko M, Zubko E, Zuilen K, Meyer P, Day A. 2004. Stable transformation of petunia
plastids. Transgenic Research 13, 523–530.
Table 1. Enzymes, biomaterials, vaccine antigens, and agronomic traits engineered via
transformation of the higher plant chloroplast genome
Enzymes/protein/trait Gene Host plant Expression
observed Reference
Biopharmaceuticals
α1-antitrypsin SERPINA1 Tobacco 2% TSP Nadai et al. (2009)
Aprotinin APR Tobacco 0.5%TSP Tissot et al. (2008)
Basic fibroblast growth factor
(bFGF)
bFGF Tobacco 0.1% TSP Wang et al. (2016)
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Bacterial phage lytic protein plyGBS Tobacco >70% TSP Oey et al. (2009a)
Cardiotrophin-1 rhct1 Tobacco 5% TSP Farran et al. (2008)
Coagulation factor IX CTB-FIX Tobacco 3.8% TSP Verma et al.
(2010b)
Cyanovirin-N CV-N fused with
GFP or PlyGBS
Tobacco Up to 0.3% TSP Elghabi et al.
(2011)
Exendin-4 (EX4) CTB-EX4 Tobacco 14.3% TLP Kwon et al. (2013)
Endolysin Cpl-1 cpl-1 Tobacco 10%TSP Oey et al. (2009b)
Endolysin Pal pal Tobacco 20% TSP Oey et al. (2009b)
Human granulocyte colony-
stimulating factor
hG-CSF Lettuce ND Sharifi Tabar et al.
(2013)
Human proinsulin CTB-Pins Tobacco
Lettuce
16% TSP
2.5% TSP
Ruhlman et al.
(2007)
Pins-Protein A Tobacco 0.2% TSP Yarbakht et al.
(2015)
Human serum albumin hsa Tobacco 11% TSP Fernandez-San
Millan et al. (2003)
Human somatotropin hST Tobacco 7% TSP Staub et al. (2000)
IFN-γ GUS-IFN-γ Tobacco 6% TSP Leelavathi and
Reddy (2003)
Insulin-like growth factor IGF-1n
IGF-1s
Tobacco 32% TSP Ruiz (2002)
Interferon-α2b (IFN-α2b) IFN-α2b Tobacco 21% TSP Arlen et al. (2007)
Thioredoxin 1 hTrx1 Lettuce 1% TSP Lim et al. (2011)
Enzymes/biomaterials
Cellulases bgl1C, cel6B, cel9A,
xeg74
Tobacco 5–40% TSP Petersen and Bock
(2011)
CelA, CelB Tobacco 22–23 mg g-1 of
TSP, 15–21 mg
g-1 of TSP
Espinoza-Sánchez
et al. (2016)
Cel6, Cel7, EndoV,
CelKI, Cel3, TF6A,
Pga2, Vlp2
peroxidase
Tobacco ND Longoni et al.
(2015)
Elastin-derived polymer eg121 Tobacco ND Guda et al. (2000)
Endo-1,4-Beta-glucanase celA Tobacco 10.7% TSP Gray et al. (2009)
Exo-cellobiohydrolase celB Tobacco 3% TSP Yu et al. (2007)
CelB Tobacco ND Longoni et al.
(2015)
Page 51
Fibronectin extra domain A EDA Tobacco 2% TCP Farran et al. (2010)
Monellin monellin Tobacco 2.5% TSP Roh et al. (2006)
p-Hydroxybenzoic acid ubiC Tobacco 13–18% TSP Viitanen (2004)
Polyhydroxybutyrate phb operon Tobacco 18.8% DW Bohmert-Tatarev et
al. (2011)
Trp asa2 Tobacco ND Zhang et al. (2001)
Xylanase xynA Tobacco 6% TSP Leelavathi et al.
(2003)
xyn Tobacco 35% TSP Castiglia et al.
(2016)
β-glucosidase Bgl1 Tobacco ND Jin et al. (2011)
Bgl1 Tobacco 20 mg g-1 of TSP Espinoza-Sánchez
et al. (2016)
celB Tobacco 60–70% TSP Castiglia et al.
(2016)
Endo-glucanase endo Tobacco ≤2% TSP Castiglia et al.
(2016)
Pectin lyase PelA Tobacco ND Espinoza-Sánchez
et al. (2015) Manganese peroxidase MnP-2 Tobacco ND
Superoxide dismutase Cu/Zn SOD Tobacco 9% TSP Madanala et al.
(2015)
Vaccine antigens (since 2012; see Lössl and Waheed, 2011 for earlier reports)
Bacterial
Anthrax protective antigen pa Tobacco 2.5–4% TSP Gorantala et al.
(2014)
Dengue virus DENV-1,2,3,4 Lettuce ND Maldaner et al.
(2013)
EDIII Tobacco 0.8–1.6% TSP Gottschamel et al.
(2016)
Haemorrhagic colitis EIT Tobacco 1.4% TSP Karimi et al. (2013)
Lyme disease OspA:YFP Tobacco 7% TSP Michoux et al.
(2013)
Pompe disease CTB-GAA Tobacco 0.1–0.2 TLP Su et al. (2015)
Porcine post-weaning diarrhoea
(PWD)
FaeG Tobacco 1% DW Kolotilin et al.
(2012)
Toxoplasmosis GRA4 Tobacco 6 μg g–1 FW Yácono et al.
(2012)
SAG1
LiHsp83-SAG1
Tobacco
Tobacco
0.1–0.2 μg g–1
FW
50–100 μg g–1
FW
Albarracín et al.
(2015)
Albarracín et al.
(2015)
Page 52
Tuberculosis antigens CTB-ESAT6 CTB-
Mtb72F
Tobacco 7.5% TSP
1.2% TSP
Lakshmi et al.
(2013)
CTB-ESAT6 Lettuce 0.75% TSP Lakshmi et al.
(2013)
Viral
Human papillomavirus GUS-E7 Tobacco 3–4% TSP Morgenfeld et al.
(2014)
HPV16 L1 Tobacco Up to 2.5% TSP Zahin et al. (2016)
Poliovirus CTB-VP1 Tobacco Native: 0.1%
TSP, Codon
optimized: 4–5%
TSP
Chan et al. (2016)
HIV/AIDS gp120, gp41 Tobacco 16 μg g–1 FW Rosales-Mendoza
et al. (2014)
HIV c4v3 Tobacco 25 μg g–1 FW Rubio-Infante et al.
(2012)
Rotavirus Vp6 Tobacco 15% TLP Inka Borchers et al.
(2012)
Agronomic Traits
Abiotic stress tolerance γ-TMT Tobacco 7.7% TLP Jin and Daniell
(2014)
Aphid/whitefly resistance Bgl-1 Tobacco ND Jin et al. (2011)
Antiviral/antibacterial/phloem-
feeding insects
pta Tobacco 9.2% TSP Jin et al. (2012)
Antiviral/antimicrobial RC101
PG1
Tobacco 32–38% TSP
17–26% TSP
Lee et al. (2011)
Cytoplasmic male sterility phaA Tobacco ND Ruiz and Daniell
(2005)
Bacterial/fungal resistance msi-99 Tobacco ND DeGray et al.
(2001)
chloroperoxidase Tobacco 10–15 μg ml–1
leaf extract
Ruhlman et al.
(2014)
msi-99 Tobacco ND Wang et al. (2015)
Drought tolerance tps1 (yeast) Tobacco ND Lee et al. (2003)
ArDH Tobacco ND Khan et al. (2015)
Herbicide resistance aroA Tobacco ND Daniell et al.
(1998)
bar Tobacco ND Iamtham and Day
(2000)
crtY Tomato/Tobacco ND Wurbs et al. (2007)
dao Tobacco ND Gisby et al. (2012)
Page 53
Insect resistance cry1A(c) Tobacco 5% TSP McBride et al.
(1995)
cry2Aa2 Tobacco 3% TSP Kota (1999)
cry2Aa2 Operon Tobacco 45.3% TSP De Cosa et al.
(2001)
cry1Aa10 Oilseed rape ND Hou et al. (2003)
cry1Ab Soybean ND Dufourmantel et al.
(2005)
cry9Aa2 Tobacco 10% TSP Chakrabarti et al.
(2006)
dsRNA of p450
monooxygenase, V-
ATPase and chitin
synthase-coding
genes
Tobacco (against
Helicoverpa
armigera)
ND Jin et al. (2015)
dsRNA of CPB,
ACT and SHR genes
Tobacco (against
Colorado potato
beetle
(Leptinotarsa
decemlineata)
ND Zhang et al. (2015)
cry1Ab Cabbage 4.8–11.1% TSP Liu et al. (2008)
Multiple biotic and abiotic
stresses
Simultaneous
expression of
protease inhibitors
and chitinase
Tobacco ND Chen et al. (2014)
Oxidative stress resistance Trx m Tobacco ND Rey et al. (2013)
Altered photosynthesis Cyanobacterial
Rubisco along with
assembly factors
RbcX, CcM35
replaced
endogenous
Rubisco large
subunit in tobacco
chloroplasts
Tobacco 12–18% Rubisco
of WT level
Lin et al. (2014)
Cyanobacterial
Rubisco without
RbcX or CcM35
Tobacco 10× lower
Rubisco than
WT
Occhialini et al.
(2016)
Page 54
replaced tobacco
large subunit
Phytoremediation merA/merB Tobacco ND Hussein et al.
(2007); Ruiz et al.
(2003)
mt1 Tobacco ND Ruiz et al. (2011)
Salt tolerance badh Carrot ND Kumar et al.
(2004a)
ArDH Tobacco ND Khan et al. (2015)
Abbreviations: TCP, total cellular proteins; TLP, total leaf proteins; TSP, total soluble proteins; ND, not
determined; DW, dry weight; FW, fresh weight (all concentrations in w/w).
Table 2. List of plants in which chloroplast transformation has been achieved
Crop Protein/trait Gene Referencea
Alfalfa Aminoglycoside adenylyl transferase,
GFP
aadA, gfp Wei et al. (2011)
Arabidopsis Aminoglycoside adenylyl transferase aadA Sikdar et al. (1998)
Cabbage Aminoglycoside adenylyl transferase aadA, uidA Liu et al. (2007)
Carrot Aminoglycoside adenylyl transferase,
Betaine aldehyde
aadA, badh Kumar et al. (2004a)
Cauliflower Aminoglycoside adenylyl transferase aadA Nugent et al. (2006)
Cotton Aminoglycoside transferase,
Neomycin phosphotransferase II
aphA6, nptII Kumar et al. (2004b)
Eggplant Aminoglycoside adenylyl transferase aadA Singh et al. (2010)
Lettuce Aminoglycoside adenylyl transferase,
GFP
aadA, gfp Lelivelt et al. (2005)
Oilseed rape Aminoglycoside adenylyl transferase,
Crystal protein insecticidal
aadA, cry1Aa10 Hou et al. (2003)
Petunia Aminoglycoside adenylyl transferase,
β-Glucuronidase
aadA, uidA Zubko et al. (2004)
Poplar Fusion protein aadA/gfp Okumura et al. (2006)
Potato Aminoglycoside adenylyl transferase,
GFP
aadA, gfp Sidorov et al. (1999)
Rice Aminoglycoside adenylyl transferase,
GFP
aadA, gfp Lee et al. (2006)
Soybean Aminoglycoside adenylyl transferase aadA Dufourmantel et al. (2004)
Sugar beet Aminoglycoside adenylyl transferase, aadA, gfp De Marchis et al. (2008)
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GFP
Sugarcane
Tobacco Aminoglycoside adenylyl transferase aadA Svab et al. (1990a)
Tomato Aminoglycoside adenylyl transferase aadA Ruf et al. (2001)
a Only the first report is included.
Table 3. Metabolic engineering in plants using plastid transformation technology
Trait engineered Genes expressed Target site Citation
Artemisinin production Twelve genes were incorporated in tobacco
plastid genome to produce isopentenyl
pyrophosphate by an engineered mevalonate
pathway for the biosynthesis of artemisinic
acid (a precursor of artemisinin)
trnI – trnA Saxena et al. (2014)
Core enzymes involved in artemisinin
biosynthesis (FPS, ADS, CYP and CPR)
were expressed first, and the promising line
was supertransformed with genes coding for
accessory enzymes (CYB5, ADH1, ALDH1,
DBR2, DXR) to increase the expression of
artemisinic acid
trnfM – trnG Fuentes et al. (2016)
Astaxanthin Three genes, CrtW, coding for b-carotene
ketolase), CrtZ, coding for b-carotene
hydroxylase, and Idi, coding for isopentenyl
diphosphate isomerase, from marine bacteria
were expressed in lettuce chloroplasts; total
carotenoid accumulation reached 230 µg g−1
fresh weight (~ 95% carotenoids)
rbcL – accD Harada et al. (2014)
β-carotene/provitamin
A
Four different bacterial and fungal genes
were expressed in in tomato fruit; provitamin
A content increased fourfold
trnfM – trnG Wurbs et al. (2007)
Lycopene β-cyclase gene from daffodil were
expressed in tomato fruits; carotenoid
accumulation was increased 50%
trnfM – trnG Apel and Bock (2009)
Dhurrin pathway Three genes coding for two membrane-bound
cytochrome P450 enzymes (CYP79A1 and
trnfM – trnG Gnanasekaran et al.
(2016)
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CYP71E1) and a soluble glucosyltransferase
(UGT85B1) were expressed in tobacco
chloroplasts to produce dhurrin, a bioactive
compound
Luciferase pathway Complete lux operon containing six genes
from Photobacterium leiognathi was
expressed in tobacco
rps12 – TrnV
trnI – trnA
Krichevsky et al.
(2010)
Mevalonate pathway Six cytosolic genes coding for the
cytoplasmic mevalonate pathway were
expressed in tobacco
trnI – trnA Kumar et al. (2012)
Engineering Rubiscoa Cyanobacterial Rubisco was assembled in
tobacco by expressing Rubisco large subunit
and small subunit coding genes along with
cofactors, transplastomic lines were much
more efficient in CO2 fixation on per unit
enzyme basis at elevated CO2
atpB – accD Occhialini et al. (2016)
Co-expression of Arabidopsis Rubisco
accumulation factor 1 (RAF1) in tobacco
improved biogenesis and accumulation of
hybrid L8AS8
t Rubisco (made of the large
subunit of Arabidopsis and the small subunit
of tobacco), as well as also led to a two-fold
increase in photosynthesis compared with the
tobacco line expressing L8AS8
t Rubisco only
atpB – accD Whitney et al. (2015)
Mutation in Methanococcoides burtonii
Rubisco (E138R, and K332E) resulted in
improved photosynthesis in tobacco
compared to the line expressing the wild-type
version of Mb Rubisco
atpB – accD Wilson et al. (2016)
Polyhydroxybutyrate Three bacterial enzymes, phbA, phbB and
phbC, were expressed in tobacco
trnN – trnR Lössl et al. (2003)
Terpene pathway Two genes coding for farnesyl diphosphate
synthase (FPS) and squalene synthase (SQS)
were expressed in tobacco chloroplast;
abnormal, massive changes in transcripts
took place
trnI – trnA Pasoreck et al. (2016)
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Vitamin E Three cyanobacterial genes encoding
homogentisate phytyltransferase (HPT),
tocopherol cyclase (TCY), and γ-tocopherol
methyltransferase (TMT) were expressed in
tomato chromoplasts; tocopherol content
increased 10-fold
trnfM – trnG Lu et al. (2013)
Three Arabidopsis genes coding for
tocopherol cyclase) or γ-TMT (γ-tocopherol
methyltransferase) and TC plus TMT (TC-
TMT) as operon were expressed in lettuce
chloroplasts; tocopherol content increased
significantly
rbcL – accD Yabuta et al. (2013)
a Only recent reports are included.
Fig. 1. Schematic representation of the process of transforming the plastid genome. (A)
Basic design of a typical vector for transforming the plastid genome. Both the expression
cassette and the selection cassette are placed between the two plastid regions. These
flanking regions are taken from the wild-type plastid genome of a plant species whose
plastome is to be manipulated, to allow a crossover event take place to integrate DNA
sequences between them. Green arrows in the chloroplast expression vector represent
promoters (P) and the direction of transcription, whereas terminators (T) are indicated by
red rectangles. The untranslated regions are represented by white circles. The thin dotted
lines with arrows indicate homologous recombination. (B) Delivery of transforming
plasmids into chloroplasts in leaf cells using a particle delivery system. The plasmid
DNA is coated on the surface of the microparticles of either gold or tungsten and then
shot on to the abaxial surface of 4- to 6-week-old sterile leaves using a gene gun. The
bombarded leaves are incubated for 48 hours in the dark, cut into small discs and placed
on regeneration medium supplemented with the appropriate antibiotic and hormones.
Primary shoots generally arise within 2–3 months. (C) The process of recovering a stable
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homoplasmic transplastomic plant line. Initially, only a few copies of the plastome are
transformed, and therefore the explant contains a mixture of both transformed as well as
untransformed copies, a state known as heteroplasmy. The wild-type copies (indicated by
light-coloured ovals) are sorted out gradually by repeating two or three regeneration
cycles under selection to reach homoplasmy, a state where all copies of the plastome are
transformed (indicated by dark grey ovals). (D) Summary of commonly used promoters,
terminators, untranslated regions, and plastome insertion sites used in chloroplast
transformation. GOI, gene of interest; P, promoter; T, terminator, ptDNA, plastid DNA;
UTR, untranslated regions.