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Journal of Experimental Botany, Vol. 64, No. 3, pp. 731–742, 2013 doi:10.

1093/jxb/ers325 Advance Access

publication 16 November, 2012

Review papeR

Chloroplast transformation for engineering of

Maureen R. Hanson1,*, Benjamin N. Gray2,3 and Beth A. Ahner2
1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
2 Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA
3 Current address: Agrivida Inc., 200 Boston Ave., Ste. 3100, Medford, MA 02155, USA

* To whom correspondence should be addressed. E-mail:

Received 27 July 2012; Revised 27 September 2012; Accepted 21 October 2012

Many efforts are underway to engineer improvements in photosynthesis to meet the challenges of increasing
demands for food and fuel in rapidly changing environmental conditions. Various transgenes have been introduced
into either the nuclear or plastid genomes in attempts to increase photosynthetic efficiency. We examine the current
knowledge of the critical features that affect levels of expression of plastid transgenes and protein accumulation in
transplastomic plants, such as promoters, 5’ and 3’ untranslated regions, RNA-processing sites, translation signals
and amino acid sequences that affect protein turnover. We review the prior attempts to manipulate the properties of
ribulose1,5-bisphosphate carboxylase oxygenase (Rubisco) through plastid transformation. We illustrate how plastid
operons could be created for expression of the multiple genes needed to introduce new pathways or enzymes to
enhance photosynthetic rates or reduce photorespiration. We describe here the past accomplishments and future
prospects for manipulating plant enzymes and pathways to enhance carbon assimilation through plastid

Key words: plastid, Rubisco, transformation vector, transgene expression.

Since transformation of the plastid genome in Chlamydomonas and Maliga, 1993) or by polyethylene glycol-mediated
and tobacco became possible (Boynton et al., 1988; Svab and transformation of protoplasts (Golds et al., 1993). Plastid
Maliga, 1993), researchers have exploited the technology to transformation is achieved by homologous recombination
understand how plastid genes are regulated, to determine the between the transformation vector and the plastid genome,
function of plastid gene products, to produce large amounts of resulting in integration of the gene(s) of interest at a predictable,
particular endogenous or foreign proteins or to alter pre-determined site (Maliga, 2004). Following incorporation of
photosynthesis or metabolism of the alga or plant. The latter transforming DNA into the chloroplast, repeated rounds of
topic will be the focus of this review: the current knowledge and selection for a marker are needed before plants reach a state of
potential for altering photosynthesis and related functions in the homoplasmy, in which all wild-type plastid genomes (plastomes)
chloroplasts of vascular plants. have been replaced with plastomes carrying the introduced DNA
(Fig. 1). The most effective selectable marker has been aadA,
General features of chloroplast which encodes aminoglycoside adenyltransferase and confers
spectinomycin resistance (Day and Goldschmidt-Clermont,
transformation 2011; Maliga, 2004).
Plastid transformation is typically performed by either biolistic It is presumed that the high copy number of chloroplast
bombardment of plant tissue with a transformation vector (Svab genomes (thousands of copies per cell) relative to
732 | Hanson et al.

Abbreviations: DB, downstream box; GFP, green fluorescent protein; IEE, intercistronic expression element; LS, large subunit; NEP, nuclear-encoded
polymerase; ORF, open reading frame; PEP, plastid-encoded polymerase; PHB, polyhydroxybutyric acid; Rubisco, ribulose-1,5-bisphosphate carboxylase
oxygenase; RuBP, ribulose-1,5-bisphosphate; SBPase, sedoheptulose-1,7-bisphosphatase; SS, small subunit; UTR, untranslated region.
© The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights
reserved. For permissions, please e-mail:

Fig. 1. Plastid transformation. (A) Steps in generating tobacco transplastomic plants are illustrated. Young seedlings growing on
culture media are bombarded with gold particles and leaf slices are then placed on spectinomycin selection medium. Initial
regenerants often require a second round of selection in order to obtain homoplasmic transplastomic plants, though we have
sometimes obtained homoplasmic transgenic plants after the first round of selection. (B) A typical plastid transformation vector ptrnI-
RT is designed for transgene insertion between the plastid trnI and trnA genes of the rRNA operon in the inverted repeat of the
plastid genome. A multicloning site is included between the T7g10 5’ untranslated region (UTR) and psbA 3’ UTR for transgene
regulation, and an aadA expression cassette flanked by loxP sites is included for spectinomycin-/streptomycin-based selection of
plastid transformants. A similar vector was used for expression of Cel6A and BglC in transgenic tobacco (Gray et al., 2009, 2011).
nuclear transgenes, which are usually present as fewer than 10 Engineering of the abundance and tissue-
copies per cell, and the lack of gene silencing in plastids, make it specificity of plastid proteins
possible to express a number of foreign proteins at extremely
high levels from the plastid genome of higher plants. There are After the demonstration that a foreign protein could accumulate
many reports of foreign protein yields of 5–15% total soluble to high levels in transformed tobacco chloroplasts, much effort
protein (reviewed in Ahmad et al., 2010; Bock and Khan, 2004; has been concerned with optimizing features of the gene
Daniell, 2006; Maliga and Bock, 2011; Scotti et al., 2012) and regulatory regions or coding region to achieve maximal
some exceptional yields of 30% total soluble protein or higher expression. These experiments have led to some general concepts
(De Cosa et al., 2001; Lentz et al., 2010; Oey et al., 2009a, concerning how to control levels of expression of chloroplast
2009b). The absence of epigenetic effects and silencing results in proteins from transgenes. Gene features of importance are the
extremely reproducible and heritable protein accumulation levels promoter, the 5’ untranslated region (UTR), the downstream box,
(Dufourmantel et al., 2006), in contrast with nuclear the N-terminal amino acid sequence, the codon usage and the 3’
transformants, where protein accumulation is quite variable UTR. The expression of a transgene can also be affected by genes
among independently transformed plants and in progeny plants located upstream and downstream. Gene expression may occur
grown from the transformed plants’ seed (Yin et al., 2004). from monocistronic or polycistronic transcripts; issues
Another important advantage of plastid transformation relative to concerning translation initiation become particularly important
nuclear transformation with respect to concerns of outcrossing of for genes located on operons. Below we provide a brief review of
transgenic pollen is that plastid genomes are very rarely the state of the knowledge of design of transgenes for expression
transmitted in pollen (Ruf et al., 2007; Svab and Maliga, 2007). at desired levels within the plastid genome. This information is
As an additional safeguard against pollen-mediated transmission relevant to our later consideration of prior attempts and the future
of antibiotic resistance, techniques have been developed to create prospects for engineering of photosynthesis through chloroplast
markerfree plastid transformants (Corneille et al., 2001; Day and transformation.
Goldschmidt-Clermont, 2011; Lutz et al., 2006).
Plastid transcription is accomplished by the combined actions of
two RNA polymerases recognizing different promoters, a T7-like
single-subunit nuclear-encoded polymerase (NEP) and a
Engineering photosynthesis by chloroplast transformation | 733
bacterium-like α2ββ’ plastid-encoded polymerase (PEP). that the T7 RNA polymerase recognizes at least some NEP
Transcription in undifferentiated plastids and in nongreen tissues promoters, resulting in altered plastid gene transcription and a
is performed primarily by the NEP, resulting in the production of pale green phenotype in seedlings when the T7 RNA polymerase
rRNA and of mRNAs encoding ribosomal proteins that are is expressed constitutively (Magee et al., 2007).
included in the PEP, ultimately resulting in the accumulation of In the approaches described above, a promoter is included in
functional PEP. Many plastid promoters have both PEP and NEP the plastid transformation vector upstream of the gene of interest
transcription start sites (Allison et al., 1996; Hajdukiewicz et al., to drive its transcription. An alternate method takes advantage of
1997). the highly processive plastid RNA polymerase and inefficient
Transcription of transgenes inserted into the plastid genome is termination at plastid 3’ UTRs (Stern and Gruissem, 1987). In
typically driven by plastid promoters included in the plastid this approach, a promoterless gene is inserted into the plastid
transformation vector, usually the 16S rRNA promoter (Prrn16) genome downstream of a highly transcribed plastid gene and the
or the psbA promoter (PpsbA). Prrn contains both PEP and NEP introduced gene is transcribed as part of a polycistron along with
transcription start sites, whereas PpsbA contains only a PEP the gene(s) normally transcribed from the plastid promoter. By
transcription start site (Allison et al., 1996). carefully choosing the insertion site in the plastid genome, this
For engineering photosynthesis, promoter systems that are approach can result in high levels of mRNA and can give
active in specific tissues or in response to light should be valuable extremely high yields of protein expressed from the transgene.
for proper regulation of modified or induced genes. For example, An early description of this type of system demonstrated that a
wasteful expression of photosynthetic proteins in non-green promoterless uidA gene inserted downstream of the plastid rbcL
tissue can be avoided, or expression of particular proteins could gene resulted in approximately 4-fold higher β-glucuronidase
be confined to particular cells within the leaf, as is the case in C4 protein levels than a construct containing a heterologous
plants. A synthetic promoter system was creating by altering the ribosomal promoter inserted at the same site in the plastid
native plastid Prrn promoter to include lac operator sequences genome, despite a greatly increased concentration of
from the Escherichia coli lac operon (Muhlbauer and Koop, monocistronic uidA mRNA in the latter case (Staub and Maliga,
2005). The novel promoter was used to drive inducible 1995). A number of researchers have since used promoterless
expression of green fluorescent protein (GFP). This approach constructs taking advantage of read-through transcription from
resulted in transplastomic tobacco lines in which GFP expression the native plastid ribosomal or psbA promoters to achieve high-
was upregulated 20-fold following the spraying of isopropyl-β-d- level protein accumulation in plastid transformants,
thiogalactopyranoside onto the plants. Another strategy for demonstrating the utility of this approach (Herz et al., 2005;
inducible regulation was demonstrated by introduction of a Chakrabarti et al., 2006; Gray et al., 2009, 2011).
riboswitch that resulted in inducible expression of GFP in the
presence of theophylline (Verhounig et al., 2010). 5’ UTRs
Several hybrid transcriptional systems have been developed
that have potential to create tissue-specificity of plastid gene Because many chloroplast genes are regulated at the
expression. These systems involve the use of a promoter posttranscriptional level (Barkan, 2011; Gruissem et al., 1988;
recognized by T7 RNA polymerase or a promoter requiring the Mallory et al., 2002), the particular 5’ UTR incorporated into a
presence of a particular sigma factor not normally present in the plastid transgene may also provide regulatory control of
plastid. Nuclear transformation is performed with transplastomic expression of a protein designed to enhance photosynthesis. The
plants to introduce a plastid-targeted T7 RNA polymerase gene most commonly used 5’ UTRs are those of the plastid psbA gene,
(McBride et al., 1995) or sigma factor gene (Buhot et al., 2006), rbcL and the bacteriophage T7gene 10. This latter 5’ UTR has
regulated by an inducible promoter. The T7 RNA polymerase been incorporated into constructs that give rise to extremely high
hybrid transcription system has been used to produce levels of transgene protein expression (Kuroda and Maliga,
polyhydroxybutyric acid (PHB) in plastids (Lössl et al., 2005) 2001a; Oey et al., 2009a, 2009b; Tregoning et al., 2003). When
because constitutive PHB production resulted in male sterility plants were grown in the light, the psbA 5’ UTR was shown to
and growth (Lössl et al., 2003). By introducing a promoter greatly affect the accumulation of β-glucuronidase protein from
recognized by T7 RNA polymerase to regulate the PHB- a uidA gene that had been placed under the control of the psbA
synthesizing enzymes and an inducible nuclear-encoded, plastid- promoter and 5’ UTR. We have recently found that two
targeted T7 RNA polymerase gene, fertile plants with normal additional bacteriophage 5’ UTRs can be used on the aadA
growth characteristics were obtained (Lössl et al., 2005), and marker gene and provide sufficient expression for recovery of
only when T7 RNA polymerase production was induced was transformants, although expression levels are low (Yang et al.,
plant growth impaired. By choosing the appropriate nuclear 2012).
promoter, either T7 RNA polymerase or the necessary sigma
factor could be produced only in certain tissues or at certain Downstream boxes
stages of plant development. Disadvantages to these hybrid
transcription systems are those associated with nuclear The downstream box (DB) region, defined by the 10–15 codons
transformation discussed above. Moreover, it has been shown immediately downstream of the start codon, was first identified
734 | Hanson et al.
in E. coli (Sprengart et al., 1996). The DB region was found to Other factors affecting protein accumulation
have major effects on accumulation of foreign protein in E. coli,
Zhou et al. (2007) reported the identification of an intercistronic
acting synergistically with the Shine–Dalgarno sequences
expression element (IEE) capable of mediating efficient
upstream of the start codon to regulate protein accumulation.
processing of polycistronic RNAs to generate stable
Kuroda and Maliga (2001b) first reported that sequences like the
monocistronic transcripts, which are sometimes required for
DB region in E. coli appeared to function in tobacco chloroplasts.
proper translation (Barkan et al., 1994). This IEE, which consists
Mutational analyses revealed that the DB RNA sequence rather
of a 50-nucleotide sequence, was derived from the intergenic
than the encoded protein sequence influenced the accumulation
region between the plastid psbN and psbH genes, normally
of foreign transgenic protein (Kuroda and Maliga, 2001b).
transcribed as part of the plastid psbB polycistron. Inclusion of
Follow-up studies on the effects of the DB region on transgene
the IEE between the yfp and nptII open reading frames (ORFs) in
regulation in plastids have found major changes in protein
the plastid transformation vector resulted in the accumulation of
accumulation from a number of different transgenes and
monocistronic yfp mRNA that was translated far more efficiently
corresponding protein products (Kuroda and Maliga, 2001a; Ye
than polycistronic transcripts, resulting in the accumulation of
et al., 2001; Gray et al., 2009, 2011). When 14 amino acid fusions
YFP protein.
from the N-terminus of TetC, NPTII or GFP were fused to either
The codon usage of foreign genes to be expressed from the
an endoglucanase gene or a β-glucosidase gene from
plastid genome should be considered in designing plastid
Thermobifida fusca, the accumulation of the enzymes varied over
transgenes. Higher plant plastid genomes are generally AT-rich,
two orders of magnitude, depending on the particular DB
which could pose a problem for expression of GC-rich foreign
sequence (Gray et al., 2009). While the TetC DB was optimal for
genes. A number of foreign genes have been altered for plastid
the endoglucanase, NPTII DB was more effective with the β-
expression from their native GC-rich coding sequences to a more
glucosidase gene. These results indicate that which DB sequence
AT-rich ORF encoding the same polypeptide, resulting in
to use to optimize expression must presently be selected
approximately 1.5–2-fold gains in protein accumulation,
empirically, as different outcomes may occur depending on the
regardless of the protein accumulation level. This is generally
particular coding region that is under its control.
less improvement than has been observed in E. coli, suggesting
that the plastid genome is better able to express ORFs not
3’ UTRs containing its preferred set of codons than E. coli (Daniell et al.,
Plastid 3’ UTRs, located immediately downstream of the stop 2009).
codon, typically contain hairpin-loop structures that facilitate
RNA maturation and processing and prevent degradation of the
RNA by ribonucleases (Monde et al., 2000b; Stern et al., 2010). Probing photosynthesis through
3’ UTRs can also affect 3’-end processing and translation chloroplast gene deletion and mutagenesis
efficiency of some genes (Eibl et al., 1999; Monde et al., 2000a).
Because of the natural homologous recombination that occurs in
3’ UTRs used to regulate foreign genes in plastids are typically
chloroplasts, vectors can be constructed to delete or mutate every
derived from plastid genes, with the rps16, rbcL, psbA and rpl32
region of the chloroplast genome. Using this strategy, the Bock
3’ UTRs being used commonly. Like DB sequences, at present
group (Bock and Khan, 2004) and others have identified the
the 3’ UTRs must be empirically selected.
function of various chloroplast ORFs, as well as determined
which ones are essential for photosynthesis. For example,
Protein stability knockout of three chloroplast genomeencoded subunits of
Until recently, little was known about the features of chloroplast photosystem I have revealed their roles in the operation,
proteins that determine their stability (De Marchis et al., 2012). assembly and stability of the complex (Krech et al., 2012;
Using a variety of N-terminal and C-terminal fusions to a gfp Schottler et al., 2011). Another complex that has been subjected
coding region in a series of 30 transgene constructs, Apel et al. to mutational analysis in transplastomic plants is chloroplast
(2010) were able to determine that the penultimate amino acid NADH dehydrogenase, implicating it in cyclic electron flow
affects protein stability, as well as some N-terminal fusions of around photosystem I (Horvath et al., 2000).
eight or nine amino acids. N-terminal fusions improve expression
of an HIV fusion inhibitor that had been difficult to express
otherwise (Elghabi et al., 2011). The coding region of the first 8 Altering vascular plant Rubisco through
to 15 N-terminal amino acids is clearly an important feature of a plastid transformation
plastid transgene that is likely to affect its level of expression.
A number of attempts have been made to engineer the tobacco
plastid genome to express foreign or mutated ribulose-1,5-
bisphosphate carboxylase oxygenase (Rubisco) subunits
(reviewed in Andrews and Whitney, 2003; Bock and Khan, 2004;
Engineering photosynthesis by chloroplast transformation | 735
Whitney et al., 2011a). Rubisco has been a focus of genetic spectinomycin resistance simultaneously with the rbcL6×His
engineering effort because of the possibility that more efficient construct, transplastomic plants could be obtained which carried
carbon fixation can be achieved by altering its functioning in a modified rbcL gene without a nearby antibiotic-resistance gene.
vascular plants (Long et al., 2006), especially in C3 plants that do Instead, the transplastomic plants carry a point mutation in the 16
not have the advantage of the carbon-concentrating mechanism S rRNA that was introduced into the plastome along with the new
extant in C4 plants. Strategies that are being pursued include rbcL gene. No difference in Rubisco activity was detected in the
altering the environment of Rubisco to be more CO 2-rich, transplastomic plants; the primary phenotype observed was
increasing its catalytic activity or reducing photorespiration, enhanced zinc Table 1. Engineering of Rubisco in transgenic
which results from reaction of ribulose-1,5-bisphosphate (RuBP) tobacco plants.
with O2 (Kajala et al., 2011; Peterhansel, 2011; Whitney et al., accumulation due to the chelating properties of the histidine tag
2011a; Zhu et al., 2010a). (Rumeau et al., 2004).
Because of the rapidity and simplicity of plastid transformation Transformation of heterologous Rubisco subunits into the
of Chlamydomonas, much has been learned from deliberate tobacco chloroplast genome has given mixed results. When an
mutagenesis of the rbcL coding region. Gene replacement has rbcL gene from tomato, which is in the same family as tobacco,
been exploited to understand determinants within Rubisco that replaced the tobacco rbcL gene in transplastomic tobacco, hybrid
affect enzyme kinetics, activation and CO2 versus O2 specificity. enzymes with tomato and tobacco subunits were obtained in pale-
Several review papers have detailed the abundant and still green plants that could grow photoautotrophically without extra
growing information resulting from mutation analysis in CO2 (Zhang et al., 2011). Plants with a sunflower-derived large
Chlamydomonas and cyanobacteria, as well as comparison of subunit (LS) and the tobacco small subunit (SS) formed
natural enzyme diversity in different species (Mueller-Cajar and functional enzyme but in amounts lower than wild-type and with
Whitney, 2008; Parry et al., 2003; Raines, 2006; Whitney et al., less activity. While plants were green on sucrose media, they
2011a). Here we will consider what has been learned from were pale green when grafted onto wild-type tobacco plants
altering Rubisco in transplastomic plants (Table 1). (Kanevski et al., 1999). The plants were later found to be able to
Tobacco shoots can regenerate and plantlets can survive in grow slowly with CO2 supplementation (Andrews and Whitney,
sucrose media even when the rbcL gene is deleted and replaced 2003). When the tobacco LS was replaced with the rbcL coding
with an aadA selectable marker gene (Kanevski and Maliga, region from Synechococcus PCC6301, the resultant plants were
1994). These pale-green plants lacking plastid rbcL were yellow and required sucrose for growth in culture medium
transformed again with a nuclear transgene comprised of the rbcL (Kanevski et al., 1999). No accumulation of the cyanobacterial
coding region fused with a chloroplast transit sequence, resulting LS or the tobacco SS was detectable, and the LS mRNA
in several plants carrying the nuclear-encoded rbcL gene that accumulated to only 10% of the abundance of the wild-type LS
exhibited 3% of normal Rubisco activity and were green while operon (Kanevski et al., 1999).
on sucrose media, but pale green while growing in low light Replacement of the tobacco rbcL with the rbcM gene from the
greenhouse conditions (Kanevski and Maliga, 1994). These alpha-proteobacterium Rhodospirillum rubrum, which has a
experiments revealed that functional Rubisco can be obtained simple homodimeric form of the enzyme, resulted in
when the large subunit is nuclear-encoded, synthesized in the photoautotrophic plants that required elevated CO2 (Whitney and
cytoplasm and imported into chloroplasts. Andrews, 2001b). Impaired translation of the bacterial gene may
Functional Rubisco could also be obtained when a 6×His tag have contributed to the lower Rubisco activity in the R. rubrum-
was placed on the C-terminus of the tobacco rbcL gene. The substituted plants (Whitney and Andrews, 2003). A codon-
production of these transplastomic plants utilized a co- optimized version of the rbcM gene has been introduced into
transformation strategy that is not commonly used, but can be tobacco for use as a ‘master line cmtrL’ for further gene-
valuable if manipulation of a plastid gene is desirable without replacement experiments. Introduction of coding regions into this
proximal introduction of a selectable marker gene. By line, which also requires supplementary CO2 for growth, places
transforming with a 16 S rRNA sequence conferring them under the control of the tobacco rbcL promoter and 5’ UTR.

Alteration in Rubisco Outcome Citation

Deletion of rbcL from plastome; introduction into nucleus Pale-green plants Kanevski and Maliga, 1994
Addition of 6×His onto rbcL in plastome Improved zinc accumulation Rumeau et al., 2004
Replace tobacco rbcL with tomato rbcL Pale-green plants Zhang et al., 2011
Replace tobacco rbcL with sunflower rbcL Pale-green plants requiring elevated CO2 Andrews and Whitney,
2003; Kanevski et al., 1999
Replace tobacco rbcL with Synechococcus rbcL Yellow plants requiring sucrose Kanevski et al., 1999
Replace tobacco rbcL with rbcM from Rhodospirillum rubrum Plants requiring elevated CO2 Whitney and Andrews,
Replace tobacco rbcL with Rubisco from Methanococcoides burtonii Plants requiring elevated CO2 Alonso et al., 2009
Replace tobacco rbcL with rbcL from C3 Flaveria Pale-green plants requiring elevated CO2 Whitney et al., 2011b
Replace tobacco rbcL with rbcL from C3–C4 intermediate or C4 Flaveria Plants grow slower than wild type in air Whitney et al., 2011b
Introduce tobacco RbcS with 6×His tag into tobacco plastome Very low expression of plastome-encoded protein Whitney and Andrews,
Introduce diatom and rhodophyte Rubisco genes into tobacco plastome Insoluble protein Whitney et al., 2001
Replace tobacco rbcL with gene encoding linked Synechococcus large Juvenile plants required elevated CO2 Whitney et al., 2009
and small subunits
736 | Hanson et al.
These plants were shown to give rise relatively rapidly to amounts exceed what is needed for maximal photosynthesis
homoplasmic lines when transformed by biolistics with (Theobald et al., 1998). Increasing the amount of Rubisco in rice
constructs carrying variant tobacco rbcL with either of two by introducing additional rbcS genes did not improve
single-amino acid changes, or rbcL fused to a ubiquitin-rbcS photosynthesis (Suzuki et al., 2007). An excess amount of
coding region (Whitney and Sharwood, 2008). Further use of this Rubisco in wild-type plants is also suggested by the phenotypes
master line for replacment of tobacco rbcL with the homologous of plants which are expressing various foreign proteins at high
gene from an archaeabacterium resulted in functional enzyme level, which has resulted in decrease in the amount of Rubisco
sufficient to support tobacco growth in enhanced CO2. The large (Bally et al., 2009, 2011). Despite the reduced amount of
subunit from this bacterium formed active decamers that Rubisco, many such plants exhibit apparently normal phenotypes
accumulated to 8–10% of total soluble protein (Alonso et al., in greenhouse and growth chamber conditions. However,
2009). The tobacco master line cmtrL has also been used to studying Rubisco-depleted plants in a greater range of conditions
introduce rbcL genes from three different Flaveria species, ones will be necessary to determine the true effect of reduced Rubisco
representing C3, a C3– C4 intermediate and C4 plants. Whereas depletion on plant growth, photosynthesis and stress tolerance. In
homoplasmic plants containing the C3–C4 and C4 species’ rbcL contrast to the results with plants in which Rubisco expression
gene could grow to maturity in air, the plants containing the rbcL was reduced by overexpression of a foreign protein, the rate of
gene from the C3 plant grew well only in supplementary CO2. CO2 assimilation was found to be limited by the amount of
Comparative analysis of the Flaveria sequences suggested some Rubisco in plants containing only a third of the normal amount
residues to target to identify their role in converting C3 to C4 of enzyme due to expression of an rbcS antisense construct in the
catalysis, leading to the identification of a methionine to nucleus (von Caemmerer et al., 1994).
isoleucine substitution as a key change leading to a C4-type
enzymatic properties (Whitney et al., 2011b).
A tobacco rbcS coding region with a 6×His tag and with or Potential for engineering of photosynthesis
without its normal transit sequence, regulated by the psbA by expression of other proteins from the
promoter, 5’ UTR and terminator, could be expressed in tobacco
transplastomic plants (Whitney and Andrews, 2001a). However,
only 1% or less of the small subunit in the Rubisco enzyme Given the large amounts of Rubisco needed in the chloroplast for
contained the 6×His tag, reflecting its plastome origin. Because photosynthesis, direct placement and optimized synthesis in the
abundant mRNA was detected, the lack of accumulation of the plastome is clearly needed for sufficient expression of the
plastome-encoded rbcS could be due to impaired translation, enzyme. Other enzymes and co-factors influencing
reduced assembly that triggers degradation or other problems photosynthesis may not be required in large amount but that
with protein stability (Whitney and Andrews, 2001a). The same should not preclude the use of plastomic insertions for their
gene-regulatory sequences were used to express the Rubisco expression given the numerous advantages of plastid
operon from a diatom and rhodophyte by insertion into a different engineering. Much of the recent effort in chloroplast
plastome location, rather than replacing the endogenous tobacco transformation technology has been to increase levels of valuable
rbcL (Whitney et al., 2001). While the foreign subunits could industrial enzymes or pharmaceuticals. However, relatively poor
accumulate to high levels, they were found in the insoluble expression—at the levels typical for nuclear transgenes or less—
fraction, indicating improper assembly resulting in aggregation. has often been observed (Maliga, 2004; Ruhlman et al., 2010).
After determining that functional Rubisco could assemble in Although undesirable when the goal is high-level protein
E. coli when the large and small subunits of Synechococcus production, low-level expression from plastid transgenes may be
PCC6301 were connected with a 40-amino acid linker (Sharwood required not to perturb metabolism or impair instead of stimulate
et al., 2008), Whitney et al. (2009) attempted a similar strategy photosynthesis. Selection of such features as 5’ UTRs,
with tobacco rbcL and rbcS. The linked tobacco subunits were downstream boxes and protein stability determinants can be
expressed from an rrn16 promoter and T7gene 10 5’ UTR made, sometimes empirically, for deliberate expression of
transgene that replaced the endogenous rbcL gene. While proteins at low instead of high levels. Furthermore, proteins
homoplasmic tobacco plants required supplementary CO 2 as constituting a novel pathway can be incorporated into an operon;
juvenile plants, reduced Rubisco activity was not primarily due current information about processing and translational initiation
to catalytic impairment, but instead to reduced accumulation of signals can aid design of vectors (Drechsel and Bock, 2011; Zhou
the assembled functional enzyme. About 30–50% of the fused et al., 2007).
Rubisco subunits were found as insoluble aggregates. The Two general strategies have been used to improve
authors pointed out that translational issues could also be photosynthesis through nuclear transgenic expression. Either
affecting the accumulation and proper folding of the linked enzymes have been expressed that have potential direct effects
subunits (Whitney et al., 2009). on photosynthetic reactions, or enzymes have been used to reduce
How much Rubisco is required for optimal growth of plants the energy loss in photorespiration (Table 2). For example, the
has been investigated, but different conclusions have been drawn enzyme sedoheptulose-1,7-bisphosphatase (SBPase), which
by different investigators. Studies in wheat indicate that Rubisco affects whether RuBP is regenerated or whether carbon exits the
Engineering photosynthesis by chloroplast transformation | 737
cycle for biosynthetic reactions, has been targeted for of two CO2 molecules that can then be utilized by Rubisco (Maier
overexpression in tobacco and rice, with improved biomass et al., 2012). Nuclear constructs were made that targeted plant
accumulation or reduced heat sensitivity (Feng et al., 2007; malate dehydrogenase and glycolate oxidase proteins into the
Lefebvre et al., 2005; Rosenthal et al., 2011; Yabuta et al., 2008). chloroplasts, converting glycolate to malate (Fahnenstich et al.,
Mixed results have been obtained by manipulation of Rubisco 2008). Because H2O2 is generated by the reaction catalysed by
activase, which is required to release inhibitory sugar phosphates glycolate oxidase, an E. coli catalase gene was also targeted to
from Rubisco to allow the carbamylation cycle to chloroplasts to remove the harmful product. Both bypasses
Table 2. Enzymes with potential for improving photosynthesis through chloroplast transgenic expression. None of these enzymes
have yet been incorporated into plastid genomes, though some have been overexpressed by nuclear transformation.
Enzymes for increasing photosynthesis References

Sedopheptalose-1,7-bisophosphatase (SBPase) Feng et al., 2007; Lefebvre et al., 2005; Rosenthal et al.,
2011; Yabuta et al., 2008

Fructose-bisphosphate aldolase (FBP aldolase) Uematsu et al., 2012; Zhu et al., 2007
ADP-glucose pyrophosphorylase (ADPGPP) Zhu et al., 2007
UDP-glucose phosphorylase Zhu et al., 2007
Bicarbonate transporters Price et al., 2011
Rubisco activase Fukayama et al., 2012; Kurek et al., 2007
Enzymes for reduction of photorespiration
Bacterial glycolate to glycerate pathway (five proteins) Kebeish et al., 2007
Glycolate catabolic pathway (glycolate oxidase (GO), malate synthase (MS), and catalase (CAT) Fahnenstich et al., 2008; Maier et al., 2012
Cyanobacterial ictB gene Lieman-Hurwitz et al., 2003

proceed. A thermostable activase mutant improves resulted in improved biomass accumulation under particular
photosynthesis and growth of Arabidopsis (Kebeish et al., 2007), growth conditions. In order to generate transgenic plants with
but overexpression of activase was not beneficial in rice (Kurek multiple genes integrated at random location in the nuclear
et al., 2007). genome, a complex series of transformation experiments and
Modelling the effect of changing the amount of enzymes of crosses had to be performed. Both of these multi-enzyme
photosynthetic carbon metabolism has indicated that increasing pathways could potentially be introduced into the chloroplast by
the expression of five enzymes, including SBPase, could possibly plastid transformation with a single operon expressing a
increase the rate of light-saturated photosynthesis. The four other polycistronic transcript with IEEs and appropriate gene
enzymes of interest from the modeling are Rubisco, fructose- regulatory signals (Khan, 2007).
bisphosphate aldolase (FBP aldolase), ADP-glucose Major efforts are underway to introduce the C4 photosynthetic
pyrophosphorylase (ADPGPP) and UDPglucose phosphorylase pathway into C3 plants such as rice (Covshoff and Hibberd, 2012;
(Zhu et al., 2007). Engineering overexpression of Rubisco would Ruan et al., 2012; Zhu et al., 2010b). Obtaining the two-cell C4
require signals for high-level expression and inclusion of both the pathway in C3 plants is challenging due to the necessity of
small and large subunit in the chloroplast transgenic locus. altering plant anatomy as well as expressing particular enzymes
However, the other three enzymes are likely needed in far smaller in the correct cell types. Chloroplast transformation may have a
amounts and could potentially be expressed from a single role to play in C4 engineering as a way to remove Rubisco
polycistronic transcript carrying IEEs, using particular 5’ UTRs, expression from mesophyll cells through rbcL deletion. Rubisco
DB sequences or N-terminal stability signals that could result in could then be specifically expressed in bundle sheath cells
differential expression levels of the three proteins. through the use of cell-specific promoters.
Attempts have also been made to engineer plants to have In addition to the strategies described above, most of which
reduced losses in photosynthetic efficiency due to have already been tested through nuclear transformation, various
photorespiration. Photorespiration can be reduced by increasing reviews have proposed expressing other types of proteins in
the amount of CO2 in the vicinity of Rubisco or by creating a way various locations to enhance photosynthesis (Ainsworth and Ort,
to bypass the process, either by engineering a reduction in 2010; Parry et al., 2011; Peterhansel and Maurino, 2011;
Rubisco’s propensity to react with oxygen, or by introducing an Peterhansel et al., 2008; Price et al., 2011; Raines, 2011; von
alternative pathway for utilization of the photorespiratory Caemmerer and Evans, 2010). These reviews also describe
substrate glycolate. Two photorespiratory bypasses have been examples of nuclear transgene expression which failed to
engineered in Arabidopsis. One strategy was to introduce five enhance photosynthesis or were detrimental.
bacterial genes encoding a chloroplast-targeted three-enzyme
(five-gene) pathway for converting glycolate to glycerate, which
can be used to regenerate RuBP (Kebeish et al., 2007). CO2 is
released in one step of the pathway and can be recovered for use
by Rubisco. A different bypass pathway results in regeneration
738 | Hanson et al.
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Substrateinduced assembly of Methanococcoides burtonii D-
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Research on photosynthesis is supported by US National Science
Buhot L, Horvath E, Medgyesy P, Lerbs-Mache S. 2006. Hybrid
Foundation grant EF-1105584 and the Division of Chemical
transcription system for controlled plastid transgene expression. The
Sciences, Geosciences, and Biosciences, Office of Basic Energy
Plant Journal 46, 700–707.
Sciences of the US Department of Energy through grant DE-
FG02-09ER16070 to MRH. Research on foreign gene expression Cardi T, Lenzi P, Maliga P. 2010. Chloroplasts as expression
was supported by US Department of Agriculture 2007–02133 platforms for plant-produced vaccines. Expert Review of Vaccines 9,
National Research Initiative Biobased Products & Bioenergy 893–911.
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US NSF Graduate P. 2006. Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts
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