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

photosynthesis
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: mrh5@cornell.edu

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

Abstract
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
transformation.

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

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

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).
Promoters
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).
Transcription in undifferentiated plastids and in nongreen tissues
is performed primarily by the NEP, resulting in the production of
rRNA and of mRNAs encoding ribosomal proteins that are
included in the PEP, ultimately resulting in the accumulation of
functional PEP. Many plastid promoters have both PEP and NEP
transcription start sites (Allison et al., 1996; Hajdukiewicz et al.,
1997).
Transcription of transgenes inserted into the plastid genome is
typically driven by plastid promoters included in the plastid
transformation vector, usually the 16S rRNA promoter (Prrn16)
or the psbA promoter (PpsbA). Prrn contains both PEP and NEP
transcription start sites, whereas PpsbA contains only a PEP
transcription start site (Allison et al., 1996).
For engineering photosynthesis, promoter systems that are
active in specific tissues or in response to light should be valuable
for proper regulation of modified or induced genes. For example,
wasteful expression of photosynthetic proteins in non-green
tissue can be avoided, or expression of particular proteins could
be confined to particular cells within the leaf, as is the case in C4
plants. A synthetic promoter system was creating by altering the
native plastid Prrn promoter to include lac operator sequences
from the Escherichia coli lac operon (Muhlbauer and Koop,
2005). The novel promoter was used to drive inducible
expression of green fluorescent protein (GFP). This approach
resulted in transplastomic tobacco lines in which GFP expression
was upregulated 20-fold following the spraying of isopropyl-β-d-
thiogalactopyranoside onto the plants. Another strategy for
inducible regulation was demonstrated by introduction of a
riboswitch that resulted in inducible expression of GFP in the
presence of theophylline (Verhounig et al., 2010).
Several hybrid transcriptional systems have been developed
that have potential to create tissue-specificity of plastid gene
expression. These systems involve the use of a promoter
recognized by T7 RNA polymerase or a promoter requiring the
presence of a particular sigma factor not normally present in the
plastid. Nuclear transformation is performed with transplastomic
plants to introduce a plastid-targeted T7 RNA polymerase gene
(McBride et al., 1995) or sigma factor gene (Buhot et al., 2006),
regulated by an inducible promoter. The T7 RNA polymerase
hybrid transcription system has been used to produce
polyhydroxybutyric acid (PHB) in plastids (Lössl et al., 2005)
because constitutive PHB production resulted in male sterility
and growth (Lössl et al., 2003). By introducing a promoter
recognized by T7 RNA polymerase to regulate the PHB-
synthesizing enzymes and an inducible nuclear-encoded, plastid-
targeted T7 RNA polymerase gene, fertile plants with normal
growth characteristics were obtained (Lössl et al., 2005), and
only when T7 RNA polymerase production was induced was
plant growth impaired. By choosing the appropriate nuclear
promoter, either T7 RNA polymerase or the necessary sigma
factor could be produced only in certain tissues or at certain
stages of plant development. Disadvantages to these hybrid
transcription systems are those associated with nuclear
transformation discussed above. Moreover, it has been shown
that the T7 RNA polymerase recognizes at least some NEP
promoters, resulting in altered plastid gene transcription and a
pale green phenotype in seedlings when the T7 RNA polymerase
is expressed constitutively (Magee et al., 2007).
In the approaches described above, a promoter is included in
the plastid transformation vector upstream of the gene of interest
to drive its transcription. An alternate method takes advantage of
the highly processive plastid RNA polymerase and inefficient
termination at plastid 3’ UTRs (Stern and Gruissem, 1987). In
this approach, a promoterless gene is inserted into the plastid
genome downstream of a highly transcribed plastid gene and the
introduced gene is transcribed as part of a polycistron along with
the gene(s) normally transcribed from the plastid promoter. By
carefully choosing the insertion site in the plastid genome, this
approach can result in high levels of mRNA and can give
extremely high yields of protein expressed from the transgene.
An early description of this type of system demonstrated that a
promoterless uidA gene inserted downstream of the plastid rbcL
gene resulted in approximately 4-fold higher β-glucuronidase
protein levels than a construct containing a heterologous
ribosomal promoter inserted at the same site in the plastid
genome, despite a greatly increased concentration of
monocistronic uidA mRNA in the latter case (Staub and Maliga,
1995). A number of researchers have since used promoterless
constructs taking advantage of read-through transcription from
the native plastid ribosomal or psbA promoters to achieve high-
level protein accumulation in plastid transformants,
demonstrating the utility of this approach (Herz et al., 2005;
Chakrabarti et al., 2006; Gray et al., 2009, 2011).
5’ UTRs
Because many chloroplast genes are regulated at the
posttranscriptional level (Barkan, 2011; Gruissem et al., 1988;
Mallory et al., 2002), the particular 5’ UTR incorporated into a
plastid transgene may also provide regulatory control of
expression of a protein designed to enhance photosynthesis. The
most commonly used 5’ UTRs are those of the plastid psbA gene,
rbcL and the bacteriophage T7gene 10. This latter 5’ UTR has
been incorporated into constructs that give rise to extremely high
levels of transgene protein expression (Kuroda and Maliga,
2001a; Oey et al., 2009a, 2009b; Tregoning et al., 2003). When
plants were grown in the light, the psbA 5’ UTR was shown to
greatly affect the accumulation of β-glucuronidase protein from
a uidA gene that had been placed under the control of the psbA
promoter and 5’ UTR. We have recently found that two
additional bacteriophage 5’ UTRs can be used on the aadA
marker gene and provide sufficient expression for recovery of
transformants, although expression levels are low (Yang et al.,
2012).
Downstream boxes
The downstream box (DB) region, defined by the 10–15 codons
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
engineering effort because of the possibility that more efficient
carbon fixation can be achieved by altering its functioning in
vascular plants (Long et al., 2006), especially in C3 plants that do
not have the advantage of the carbon-concentrating mechanism
extant in C4 plants. Strategies that are being pursued include
altering the environment of Rubisco to be more CO 2-rich,
increasing its catalytic activity or reducing photorespiration,
which results from reaction of ribulose-1,5-bisphosphate (RuBP)
with O2 (Kajala et al., 2011; Peterhansel, 2011; Whitney et al.,
2011a; Zhu et al., 2010a).
Because of the rapidity and simplicity of plastid transformation
of Chlamydomonas, much has been learned from deliberate
mutagenesis of the rbcL coding region. Gene replacement has
been exploited to understand determinants within Rubisco that
affect enzyme kinetics, activation and CO2 versus O2 specificity.
Several review papers have detailed the abundant and still
growing information resulting from mutation analysis in
Chlamydomonas and cyanobacteria, as well as comparison of
natural enzyme diversity in different species (Mueller-Cajar and
Whitney, 2008; Parry et al., 2003; Raines, 2006; Whitney et al.,
2011a). Here we will consider what has been learned from
altering Rubisco in transplastomic plants (Table 1).
Tobacco shoots can regenerate and plantlets can survive in
sucrose media even when the rbcL gene is deleted and replaced
with an aadA selectable marker gene (Kanevski and Maliga,
1994). These pale-green plants lacking plastid rbcL were
transformed again with a nuclear transgene comprised of the rbcL
coding region fused with a chloroplast transit sequence, resulting
in several plants carrying the nuclear-encoded rbcL gene that
exhibited 3% of normal Rubisco activity and were green while
on sucrose media, but pale green while growing in low light
greenhouse conditions (Kanevski and Maliga, 1994). These
experiments revealed that functional Rubisco can be obtained
when the large subunit is nuclear-encoded, synthesized in the
cytoplasm and imported into chloroplasts.
Functional Rubisco could also be obtained when a 6×His tag
was placed on the C-terminus of the tobacco rbcL gene. The
production of these transplastomic plants utilized a co-
transformation strategy that is not commonly used, but can be
valuable if manipulation of a plastid gene is desirable without
proximal introduction of a selectable marker gene. By
transforming with a 16 S rRNA sequence conferring
Alteration in Rubisco
Deletion of rbcL from plastome; introduction into nucleus
Addition of 6×His onto rbcL in plastome
Replace tobacco rbcL with tomato rbcL
Replace tobacco rbcL with sunflower rbcL
Replace tobacco rbcL with Synechococcus rbcL
Replace tobacco rbcL with rbcM from Rhodospirillum rubrum
Replace tobacco rbcL with Rubisco from Methanococcoides burtonii
Replace tobacco rbcL with rbcL from C3 Flaveria
Replace tobacco rbcL with rbcL from C3–C4 intermediate or C4 Flaveria
Introduce tobacco RbcS with 6×His tag into tobacco plastome
Introduce diatom and rhodophyte Rubisco genes into tobacco plastome
Replace tobacco rbcL with gene encoding linked Synechococcus large
and small subunits
spectinomycin resistance simultaneously with the rbcL6×His
construct, transplastomic plants could be obtained which carried
a modified rbcL gene without a nearby antibiotic-resistance gene.
Instead, the transplastomic plants carry a point mutation in the 16
S rRNA that was introduced into the plastome along with the new
rbcL gene. No difference in Rubisco activity was detected in the
transplastomic plants; the primary phenotype observed was
enhanced zinc Table 1. Engineering of Rubisco in transgenic
tobacco plants.
accumulation due to the chelating properties of the histidine tag
(Rumeau et al., 2004).
Transformation of heterologous Rubisco subunits into the
tobacco chloroplast genome has given mixed results. When an
rbcL gene from tomato, which is in the same family as tobacco,
replaced the tobacco rbcL gene in transplastomic tobacco, hybrid
enzymes with tomato and tobacco subunits were obtained in pale-
green plants that could grow photoautotrophically without extra
CO2 (Zhang et al., 2011). Plants with a sunflower-derived large
subunit (LS) and the tobacco small subunit (SS) formed
functional enzyme but in amounts lower than wild-type and with
less activity. While plants were green on sucrose media, they
were pale green when grafted onto wild-type tobacco plants
(Kanevski et al., 1999). The plants were later found to be able to
grow slowly with CO2 supplementation (Andrews and Whitney,
2003). When the tobacco LS was replaced with the rbcL coding
region from Synechococcus PCC6301, the resultant plants were
yellow and required sucrose for growth in culture medium
(Kanevski et al., 1999). No accumulation of the cyanobacterial
LS or the tobacco SS was detectable, and the LS mRNA
accumulated to only 10% of the abundance of the wild-type LS
operon (Kanevski et al., 1999).
Replacement of the tobacco rbcL with the rbcM gene from the
alpha-proteobacterium Rhodospirillum rubrum, which has a
simple homodimeric form of the enzyme, resulted in
photoautotrophic plants that required elevated CO2 (Whitney and
Andrews, 2001b). Impaired translation of the bacterial gene may
have contributed to the lower Rubisco activity in the R. rubrum-
substituted plants (Whitney and Andrews, 2003). A codon-
optimized version of the rbcM gene has been introduced into
tobacco for use as a ‘master line cmtrL’ for further gene-
replacement experiments. Introduction of coding regions into this
line, which also requires supplementary CO2 for growth, places
them under the control of the tobacco rbcL promoter and 5’ UTR.
Outcome Citation
Pale-green plants Kanevski and Maliga, 1994
Improved zinc accumulation Rumeau et al., 2004
Pale-green plants Zhang et al., 2011
Pale-green plants requiring elevated CO2 Andrews and Whitney,
2003; Kanevski et al., 1999
Yellow plants requiring sucrose Kanevski et al., 1999
Plants requiring elevated CO2 Whitney and Andrews,
2001b
Plants requiring elevated CO2 Alonso et al., 2009
Pale-green plants requiring elevated CO2 Whitney et al., 2011b
Plants grow slower than wild type in air Whitney et al., 2011b
Very low expression of plastome-encoded protein Whitney and Andrews,
2001a
Insoluble protein Whitney et al., 2001
Juvenile plants required elevated CO2 Whitney et al., 2009
736 | Hanson et al.
These plants were shown to give rise relatively rapidly to
homoplasmic lines when transformed by biolistics with
constructs carrying variant tobacco rbcL with either of two
single-amino acid changes, or rbcL fused to a ubiquitin-rbcS
coding region (Whitney and Sharwood, 2008). Further use of this
master line for replacment of tobacco rbcL with the homologous
gene from an archaeabacterium resulted in functional enzyme
sufficient to support tobacco growth in enhanced CO2. The large
subunit from this bacterium formed active decamers that
accumulated to 8–10% of total soluble protein (Alonso et al.,
2009). The tobacco master line cmtrL has also been used to
introduce rbcL genes from three different Flaveria species, ones
representing C3, a C3– C4 intermediate and C4 plants. Whereas
homoplasmic plants containing the C3–C4 and C4 species’ rbcL
gene could grow to maturity in air, the plants containing the rbcL
gene from the C3 plant grew well only in supplementary CO2.
Comparative analysis of the Flaveria sequences suggested some
residues to target to identify their role in converting C3 to C4
catalysis, leading to the identification of a methionine to
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
without its normal transit sequence, regulated by the psbA
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
contained the 6×His tag, reflecting its plastome origin. Because
abundant mRNA was detected, the lack of accumulation of the
plastome-encoded rbcS could be due to impaired translation,
reduced assembly that triggers degradation or other problems
with protein stability (Whitney and Andrews, 2001a). The same
gene-regulatory sequences were used to express the Rubisco
operon from a diatom and rhodophyte by insertion into a different
plastome location, rather than replacing the endogenous tobacco
rbcL (Whitney et al., 2001). While the foreign subunits could
accumulate to high levels, they were found in the insoluble
fraction, indicating improper assembly resulting in aggregation.
After determining that functional Rubisco could assemble in
E. coli when the large and small subunits of Synechococcus
PCC6301 were connected with a 40-amino acid linker (Sharwood
et al., 2008), Whitney et al. (2009) attempted a similar strategy
with tobacco rbcL and rbcS. The linked tobacco subunits were
expressed from an rrn16 promoter and T7gene 10 5’ UTR
transgene that replaced the endogenous rbcL gene. While
homoplasmic tobacco plants required supplementary CO 2 as
juvenile plants, reduced Rubisco activity was not primarily due
to catalytic impairment, but instead to reduced accumulation of
the assembled functional enzyme. About 30–50% of the fused
Rubisco subunits were found as insoluble aggregates. The
authors pointed out that translational issues could also be
affecting the accumulation and proper folding of the linked
subunits (Whitney et al., 2009).
How much Rubisco is required for optimal growth of plants
has been investigated, but different conclusions have been drawn
by different investigators. Studies in wheat indicate that Rubisco
amounts exceed what is needed for maximal photosynthesis
(Theobald et al., 1998). Increasing the amount of Rubisco in rice
by introducing additional rbcS genes did not improve
photosynthesis (Suzuki et al., 2007). An excess amount of
Rubisco in wild-type plants is also suggested by the phenotypes
of plants which are expressing various foreign proteins at high
level, which has resulted in decrease in the amount of Rubisco
(Bally et al., 2009, 2011). Despite the reduced amount of
Rubisco, many such plants exhibit apparently normal phenotypes
in greenhouse and growth chamber conditions. However,
studying Rubisco-depleted plants in a greater range of conditions
will be necessary to determine the true effect of reduced Rubisco
depletion on plant growth, photosynthesis and stress tolerance. In
contrast to the results with plants in which Rubisco expression
was reduced by overexpression of a foreign protein, the rate of
CO2 assimilation was found to be limited by the amount of
Rubisco in plants containing only a third of the normal amount
of enzyme due to expression of an rbcS antisense construct in the
nucleus (von Caemmerer et al., 1994).
Potential for engineering of photosynthesis
by expression of other proteins from the
plastome
Given the large amounts of Rubisco needed in the chloroplast for
photosynthesis, direct placement and optimized synthesis in the
plastome is clearly needed for sufficient expression of the
enzyme. Other enzymes and co-factors influencing
photosynthesis may not be required in large amount but that
should not preclude the use of plastomic insertions for their
expression given the numerous advantages of plastid
engineering. Much of the recent effort in chloroplast
transformation technology has been to increase levels of valuable
industrial enzymes or pharmaceuticals. However, relatively poor
expression—at the levels typical for nuclear transgenes or less—
has often been observed (Maliga, 2004; Ruhlman et al., 2010).
Although undesirable when the goal is high-level protein
production, low-level expression from plastid transgenes may be
required not to perturb metabolism or impair instead of stimulate
photosynthesis. Selection of such features as 5’ UTRs,
downstream boxes and protein stability determinants can be
made, sometimes empirically, for deliberate expression of
proteins at low instead of high levels. Furthermore, proteins
constituting a novel pathway can be incorporated into an operon;
current information about processing and translational initiation
signals can aid design of vectors (Drechsel and Bock, 2011; Zhou
et al., 2007).
Two general strategies have been used to improve
photosynthesis through nuclear transgenic expression. Either
enzymes have been expressed that have potential direct effects
on photosynthetic reactions, or enzymes have been used to reduce
the energy loss in photorespiration (Table 2). For example, the
enzyme sedoheptulose-1,7-bisphosphatase (SBPase), which
affects whether RuBP is regenerated or whether carbon exits the
 Engineering photosynthesis by chloroplast transformation | 737
cycle for biosynthetic reactions, has been targeted for
overexpression in tobacco and rice, with improved biomass
accumulation or reduced heat sensitivity (Feng et al., 2007;
Lefebvre et al., 2005; Rosenthal et al., 2011; Yabuta et al., 2008).
Mixed results have been obtained by manipulation of Rubisco
activase, which is required to release inhibitory sugar phosphates
from Rubisco to allow the carbamylation cycle to
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
Sedopheptalose-1,7-bisophosphatase (SBPase)
Fructose-bisphosphate aldolase (FBP aldolase)
ADP-glucose pyrophosphorylase (ADPGPP)
UDP-glucose phosphorylase
Bicarbonate transporters
Rubisco activase
Enzymes for reduction of photorespiration
Bacterial glycolate to glycerate pathway (five proteins)
Glycolate catabolic pathway (glycolate oxidase (GO), malate synthase (MS), and catalase (CAT) Fahnenstich et al., 2008; Maier et al., 2012
Cyanobacterial ictB gene
proceed. A thermostable activase mutant improves
photosynthesis and growth of Arabidopsis (Kebeish et al., 2007),
but overexpression of activase was not beneficial in rice (Kurek
et al., 2007).
Modelling the effect of changing the amount of enzymes of
photosynthetic carbon metabolism has indicated that increasing
the expression of five enzymes, including SBPase, could possibly
increase the rate of light-saturated photosynthesis. The four other
enzymes of interest from the modeling are Rubisco, fructose-
bisphosphate aldolase (FBP aldolase), ADP-glucose
pyrophosphorylase (ADPGPP) and UDPglucose phosphorylase
(Zhu et al., 2007). Engineering overexpression of Rubisco would
require signals for high-level expression and inclusion of both the
small and large subunit in the chloroplast transgenic locus.
However, the other three enzymes are likely needed in far smaller
amounts and could potentially be expressed from a single
polycistronic transcript carrying IEEs, using particular 5’ UTRs,
DB sequences or N-terminal stability signals that could result in
differential expression levels of the three proteins.
Attempts have also been made to engineer plants to have
reduced losses in photosynthetic efficiency due to
photorespiration. Photorespiration can be reduced by increasing
the amount of CO2 in the vicinity of Rubisco or by creating a way
to bypass the process, either by engineering a reduction in
Rubisco’s propensity to react with oxygen, or by introducing an
alternative pathway for utilization of the photorespiratory
substrate glycolate. Two photorespiratory bypasses have been
engineered in Arabidopsis. One strategy was to introduce five
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
of two CO2 molecules that can then be utilized by Rubisco (Maier
et al., 2012). Nuclear constructs were made that targeted plant
malate dehydrogenase and glycolate oxidase proteins into the
chloroplasts, converting glycolate to malate (Fahnenstich et al.,
2008). Because H2O2 is generated by the reaction catalysed by
glycolate oxidase, an E. coli catalase gene was also targeted to
chloroplasts to remove the harmful product. Both bypasses
References
Feng et al., 2007; Lefebvre et al., 2005; Rosenthal et al.,
2011; Yabuta et al., 2008
Uematsu et al., 2012; Zhu et al., 2007
Zhu et al., 2007
Zhu et al., 2007
Price et al., 2011
Fukayama et al., 2012; Kurek et al., 2007
Kebeish et al., 2007
Lieman-Hurwitz et al., 2003
resulted in improved biomass accumulation under particular
growth conditions. In order to generate transgenic plants with
multiple genes integrated at random location in the nuclear
genome, a complex series of transformation experiments and
crosses had to be performed. Both of these multi-enzyme
pathways could potentially be introduced into the chloroplast by
plastid transformation with a single operon expressing a
polycistronic transcript with IEEs and appropriate gene
regulatory signals (Khan, 2007).
Major efforts are underway to introduce the C4 photosynthetic
pathway into C3 plants such as rice (Covshoff and Hibberd, 2012;
Ruan et al., 2012; Zhu et al., 2010b). Obtaining the two-cell C4
pathway in C3 plants is challenging due to the necessity of
altering plant anatomy as well as expressing particular enzymes
in the correct cell types. Chloroplast transformation may have a
role to play in C4 engineering as a way to remove Rubisco
expression from mesophyll cells through rbcL deletion. Rubisco
could then be specifically expressed in bundle sheath cells
through the use of cell-specific promoters.
In addition to the strategies described above, most of which
have already been tested through nuclear transformation, various
reviews have proposed expressing other types of proteins in
various locations to enhance photosynthesis (Ainsworth and Ort,
2010; Parry et al., 2011; Peterhansel and Maurino, 2011;
Peterhansel et al., 2008; Price et al., 2011; Raines, 2011; von
Caemmerer and Evans, 2010). These reviews also describe
examples of nuclear transgene expression which failed to
enhance photosynthesis or were detrimental.
738 | Hanson et al.
Limitations to plastid transformation
technology for improving photosynthesis
One of the major problems limiting plastid transformation for
enhancing photosynthesis in crop plants is the current inability to
obtain regenerated homoplasmic monocotyledonous
transplastomic plants (Khan, 2007; Lee et al., 2006). A recent
report of wheat plastid transformation has been retracted (Cui et
al., 2011). New selectable markers or new tissue culture
techniques are likely to be necessary to obtain transformants in
the important monocotyledonous grasses. A barrier to learning
more about photosynthesis and plastid gene regulation is the
absence of a method to obtain regenerated fertile Arabidopsis
transplastomic plants, preventing the utilization of the abundant
Arabidopsis genetic and genomic resources in conjunction with
deliberately altered plastid genes.
Plastid transformation of tobacco (Nicotiana tabacum) is now
a relatively routine procedure, and a number of other Solanaceous
species including tomato (Ruf et al., 2001), petunia (Zubkot et
al., 2004), eggplant (aubergine; Singh et al., 2010) and potato
(Sidorov et al., 1999) are transformable. A reasonable strategy is
to optimize the desired level of expression in Nicotiana and then
introduce the construct into the crop species of interest, given that
many features of chloroplast genes and genomes are highly
conserved in vascular plants. However, while gene regulation is
generally conserved, the effect on photosynthesis of expressing a
particular protein may vary between different plant species.
Recent lists of plastid-transformable species have been provided
in several reviews (Cardi et al., 2010; Day and Goldschmidt-
Clermont, 2011). Major effort was necessary to make nuclear
transformation a reality for many major crop plants and will also
likely be necessary to expand the number of important plants for
which plastid genomes can be engineered.
Acknowledgements
Research on photosynthesis is supported by US National Science
Foundation grant EF-1105584 and the Division of Chemical
Sciences, Geosciences, and Biosciences, Office of Basic Energy
Sciences of the US Department of Energy through grant DE-
FG02-09ER16070 to MRH. Research on foreign gene expression
was supported by US Department of Agriculture 2007–02133
National Research Initiative Biobased Products & Bioenergy
Production program grant to MRH and BAA. BNG received a
US NSF Graduate
Fellowship.
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