Journal of Experimental Botany Advance Access published November 16, 2012
Journal of Experimental Botany
Botany, Vol. 63, No. 2, pp. 695–709, 2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers325 Advance Access publication 4 November, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH
PAPER
Review paper
Chloroplast
for engineering
of photosynthesis
In
Posidoniatransformation
oceanica cadmium
induces changes
in DNA
methylation and chromatin patterning
Maureen R. Hanson1,*, Benjamin N. Gray2,3 and Beth A. Ahner2
1 Department
of Molecular
Biology and Genetics,
Cornell
University,
14853, USA
Maria
Greco,
Adriana Chiappetta,
Leonardo
Bruno
and Ithaca,
Maria NY
Beatrice
Bitonti*
2 Received 29
27 July
September
2012; Accepted
21 2011
October 2012
Received
May 2012;
2011; Revised
Revised 27
8 July
2011; Accepted
18 August
Abstract
Abstract
Many
efforts are
underway
to engineer
improvements
in photosynthesis
to meet
the challenges
of increasing demands
In
mammals,
cadmium
is widely
considered
as a non-genotoxic
carcinogen
acting
through a methylation-dependent
for food and
fuel in rapidly
changing
environmental
conditions.
transgenes
have are
been
introduced
into either
epigenetic
mechanism.
Here,
the effects
of Cd treatment
on theVarious
DNA methylation
patten
examined
together
with
theeffect
nuclear
plastid genomes
in attempts
to increase
photosynthetic
efficiency. We
examine
the current
knowledge
its
onorchromatin
reconfiguration
in Posidonia
oceanica.
DNA methylation
level
and pattern
were analysed
in
of the critical
features
that
affect
levels
of and
expression
plastid
transgenes
in transplasactively
growing
organs,
under
short(6 h)
long- (2 of
d or
4 d) term
and lowand
(10 protein
mM) andaccumulation
high (50 mM) doses
of Cd,
tomic plants,
such as promoters, 5’Amplification
and 3’ untranslated
regions, RNA-processing
translation signals and
amino
through
a Methylation-Sensitive
Polymorphism
technique and sites,
an immunocytological
approach,
acid sequences
affect protein
review
the prior attempts to(CMT)
manipulate
properties
of ribuloserespectively.
Thethat
expression
of oneturnover.
memberWe
of the
CHROMOMETHYLASE
family, the
a DNA
methyltransferase,
1,5-bisphosphate
carboxylase
oxygenase
(Rubisco)
through
plastid transformation.
We illustrate
how plastid electron
operons
was
also assessed
by qRT-PCR.
Nuclear
chromatin
ultrastructure
was investigated
by transmission
could be created
for expression
of athe
multiple
genes needed
introduce
new pathways
enzymes
to enhance
microscopy.
Cd treatment
induced
DNA
hypermethylation,
asto
well
as an up-regulation
of or
CMT,
indicating
that de
photosynthetic
rates
reduceoccur.
photorespiration.
describe
and future prospects
novo
methylation
didor
indeed
Moreover, aWe
high
dose ofhere
Cd the
led past
to a accomplishments
progressive heterochromatinization
of
for manipulating
enzymes and
pathways
to enhance
assimilation
throughThe
plastid
interphase
nucleiplant
and apoptotic
figures
were also
observedcarbon
after long-term
treatment.
datatransformation.
demonstrate that Cd
perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are
Key words: plastid,chromatin
Rubisco, transformation
vector,
transgene
expression.
linked
to nuclear
reconfiguration
likely
to establish
a new balance of expressed/repressed chromatin.
Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
Introduction Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
DNA-methylation,
Since transformation of the plastid genome in Chlamydomonas (Svab 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
Introduction
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
in integration
of the
interestinat aterrestrial
predictIn
the Mediterranean
coastal ecosystem,
the amounts
endemic resulting
Although
not essential
forgene(s)
plant ofgrowth,
of
particular
endogenous
or
foreign
proteins
or
to
alter
phoable,
pre-determined
site
(Maliga,
2004).
Following
incorposeagrass Posidonia oceanica (L.) Delile plays a relevant role plants, Cd is readily absorbed by roots and translocated into
tosynthesis
metabolism
of the alga
or oxygenation
plant. The latter
ration organs
of transforming
DNA into
the it
chloroplast,
repeated
by
ensuringorprimary
production,
water
and aerial
while, in acquatic
plants,
is directly taken
up
topic
will
be
the
focus
of
this
review:
the
current
knowledge
rounds
of
selection
for
a
marker
are
needed
before
plants
provides niches for some animals, besides counteracting by leaves. In plants, Cd absorption induces complex changes
and potential
altering
and related
func- at
reach
state ofbiochemical
homoplasmy,
which all wild-type
plascoastal
erosionfor
through
its photosynthesis
widespread meadows
(Ott, 1980;
the agenetic,
andin physiological
levels which
tions
in
the
chloroplasts
of
vascular
plants.
tid
genomes
(plastomes)
have
been
replaced
with
plastomes
Piazzi et al., 1999; Alcoverro et al., 2001). There is also ultimately account for its toxicity (Valle and Ulmer, 1972;
carryingdithe
introduced
DNA 1999;
(Fig. 1).
The most
effective
considerable evidence that P. oceanica plants are able to Sanitz
Toppi
and Gabrielli,
Benavides
et al.,
2005;
selectable
marker
has
been
aadA,
which
encodes
aminoglycoabsorb
andfeatures
accumulate
from sediments (Sanchiz Weber et al., 2006; Liu et al., 2008). The most obvious
General
of metals
chloroplast
side adenyltransferase
spectinomycin
resistance
et
al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus symptom
of Cd toxicityand
is a confers
reduction
in plant growth
due to
transformation
(Day
and
Goldschmidt-Clermont,
2011;
Maliga,
2004).
influencing metal bioavailability in the marine ecosystem. an inhibition of photosynthesis, respiration, and nitrogen
Plastid
typicallyis performed
by either biolisIt is presumed
that
high copy
of mineral
chloroFor
thistransformation
reason, this is
seagrass
widely considered
to be metabolism,
as well
as the
a reduction
in number
water and
tic
bombardment
of
plant
tissue
with
a
transformation
vector
plast
genomes
(thousands
of
copies
per
cell)
relative
to
a metal bioindicator species (Maserti et al., 1988; Pergent uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000;
et al., 1995; Lafabrie et al., 2007). Cd is one of most Shukla et al., 2003; Sobkowiak and Deckert, 2003).
widespread
heavy
metals
both
and IEE,
marine
the genetic
bothNEP,
animals
and plants,
Cd
Abbreviations: DB,
downstream
box;inGFP,
greenterrestrial
fluorescent protein;
intercistronicAt
expression
element; level,
LS, largeinsubunit;
nuclear-encoded
polymerase;
ORF, open reading frame; PEP, plastid-encoded polymerase; PHB, polyhydroxybutyric
Rubisco,
ribulose-1,5-bisphosphate
carboxylase
oxygenase; RuBP,
environments.
can acid;
induce
chromosomal
aberrations,
abnormalities
in
ribulose-1,5-bisphosphate; SBPase, sedoheptulose-1,7-bisphosphatase; SS, small subunit; UTR, untranslated region.
© 2011
The Author
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
ª
The Author(s).
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email: distributed
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is an Open Access
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Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA
3 Department
of Ecology,
University
of Boston
Calabria,Ave.,
Laboratory
of Plant
Cyto-physiology,
Ponte Pietro Bucci, I-87036 Arcavacata di Rende,
Current address:
Agrivida
Inc., 200
Ste. 3100,
Medford,
MA 02155, USA
Cosenza, Italy
To whom
** To
whom correspondence
correspondence should
should be
be addressed.
addressed. E-mail:
E-mail: [email protected]
[email protected]
Page 2 of 12 | Hanson et al.
nuclear transgenes, which are usually present as fewer than
10 copies per cell, and the lack of gene silencing in plastids, make it possible to express a number of foreign proteins at extremely high levels from the plastid genome of
higher plants. There are many reports of foreign protein
yields of 5–15% total soluble protein (reviewed in Ahmad
et al., 2010; Bock and Khan, 2004; Daniell, 2006; Maliga
and Bock, 2011; Scotti et al., 2012) and some exceptional
yields of 30% total soluble protein or higher (De Cosa
et al., 2001; Lentz et al., 2010; Oey et al., 2009a, 2009b).
The absence of epigenetic effects and silencing results in
extremely reproducible and heritable protein accumulation
levels (Dufourmantel et al., 2006), in contrast with nuclear
transformants, where protein accumulation is quite variable
among independently transformed plants and in progeny
plants grown from the transformed plants’ seed (Yin et al.,
2004). Another important advantage of plastid transformation relative to nuclear transformation with respect to
concerns of outcrossing of transgenic pollen is that plastid
genomes are very rarely transmitted in pollen (Ruf et al.,
2007; Svab and Maliga, 2007). As an additional safeguard
against pollen-mediated transmission of antibiotic resistance, techniques have been developed to create markerfree plastid transformants (Corneille et al., 2001; Day and
Goldschmidt-Clermont, 2011; Lutz et al., 2006).
Engineering of the abundance and
tissue-specificity of plastid proteins
After the demonstration that a foreign protein could accumulate to high levels in transformed tobacco chloroplasts,
much effort has been concerned with optimizing features of
the gene regulatory regions or coding region to achieve maximal expression. These experiments have led to some general
concepts concerning how to control levels of expression of
chloroplast proteins from transgenes. Gene features of importance are the promoter, the 5’ untranslated region (UTR), the
downstream box, the N-terminal amino acid sequence, the
codon usage and the 3’ UTR. The expression of a transgene
can also be affected by genes located upstream and downstream. Gene expression may occur from monocistronic or
polycistronic transcripts; issues concerning translation initiation become particularly important for genes located on
operons. Below we provide a brief review of the state of the
knowledge of design of transgenes for expression at desired
levels within the plastid genome. This information is relevant
to our later consideration of prior attempts and the future
prospects for engineering of photosynthesis through chloroplast transformation.
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 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).
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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).
Engineering photosynthesis by chloroplast transformation | Page 3 of 12
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 in E. coli (Sprengart et al., 1996). The DB region was
found to have major effects on accumulation of foreign protein in E. coli, acting synergistically with the Shine–Dalgarno
sequences upstream of the start codon to regulate protein
accumulation. Kuroda and Maliga (2001b) first reported
that sequences like the DB region in E. coli appeared to function in tobacco chloroplasts. Mutational analyses revealed
that the DB RNA sequence rather than the encoded protein
sequence influenced the accumulation of foreign transgenic
protein (Kuroda and Maliga, 2001b).
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
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
Page 4 of 12 | Hanson et al.
3’ UTRs
Plastid 3’ UTRs, located immediately downstream of the stop
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). 3’ UTRs can also affect 3’-end processing and
translation efficiency of some genes (Eibl et al., 1999; Monde
et al., 2000a). 3’ UTRs used to regulate foreign genes in plastids are typically derived from plastid genes, with the rps16,
rbcL, psbA and rpl32 3’ UTRs being used commonly. Like
DB sequences, at present the 3’ UTRs must be empirically
selected.
Protein stability
Until recently, little was known about the features of chloroplast proteins that determine their stability (De Marchis et al.,
2012). Using a variety of N-terminal and C-terminal fusions
to a gfp coding region in a series of 30 transgene constructs,
Apel et al. (2010) were able to determine that the penultimate amino acid affects protein stability, as well as some
N-terminal fusions of 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 to 15 N-terminal amino acids
is clearly an important feature of a plastid transgene that is
likely to affect its level of expression.
Other factors affecting protein accumulation
Zhou et al. (2007) reported the identification of an intercistronic expression element (IEE) capable of mediating efficient processing of polycistronic RNAs to generate stable
monocistronic transcripts, which are sometimes required for
proper translation (Barkan et al., 1994). This IEE, which
consists of a 50-nucleotide sequence, was derived from the
intergenic region between the plastid psbN and psbH genes,
normally transcribed as part of the plastid psbB polycistron.
Inclusion of the IEE between the yfp and nptII open reading
frames (ORFs) in the plastid transformation vector resulted
in the accumulation of monocistronic yfp mRNA that was
translated far more efficiently than polycistronic transcripts,
resulting in the accumulation of YFP protein.
The codon usage of foreign genes to be expressed from
the plastid genome should be considered in designing plastid transgenes. Higher plant plastid genomes are generally AT-rich, which could pose a problem for expression of
GC-rich foreign genes. A number of foreign genes have been
altered for plastid expression from their native GC-rich coding sequences to a more AT-rich ORF encoding the same
polypeptide, resulting in approximately 1.5–2-fold gains in
protein accumulation, regardless of the protein accumulation level. This is generally less improvement than has been
observed in E. coli, suggesting that the plastid genome is better able to express ORFs not containing its preferred set of
codons than E. coli (Daniell et al., 2009).
Probing photosynthesis through
chloroplast gene deletion and mutagenesis
Because of the natural homologous recombination that
occurs in chloroplasts, vectors can be constructed to delete
or mutate every region of the chloroplast genome. Using this
strategy, the Bock group (Bock and Khan, 2004) and others have identified the function of various chloroplast ORFs,
as well as determined which ones are essential for photosynthesis. For example, knockout of three chloroplast genomeencoded subunits of photosystem I have revealed their roles
in the operation, assembly and stability of the complex
(Krech et al., 2012; Schottler et al., 2011). Another complex
that has been subjected to mutational analysis in transplastomic plants is chloroplast NADH dehydrogenase, implicating it in cyclic electron flow around photosystem I (Horvath
et al., 2000).
Altering vascular plant Rubisco through
plastid transformation
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; 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 CO2-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
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
Follow-up studies on the effects of the DB region on
transgene regulation in plastids have found major changes in
protein accumulation from a number of different transgenes
and corresponding protein products (Kuroda and Maliga,
2001a; Ye et al., 2001; Gray et al., 2009, 2011). When 14 amino
acid fusions from the N-terminus of TetC, NPTII or GFP
were fused to either an endoglucanase gene or a β-glucosidase
gene from Thermobifida fusca, the accumulation of the
enzymes varied over two orders of magnitude, depending
on the particular DB sequence (Gray et al., 2009). While the
TetC DB was optimal for the endoglucanase, NPTII DB was
more effective with the β-glucosidase gene. These results indicate that which DB sequence to use to optimize expression
must presently be selected empirically, as different outcomes
may occur depending on the particular coding region that is
under its control.
Engineering photosynthesis by chloroplast transformation | Page 5 of 12
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. 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
Table 1. Engineering of Rubisco in transgenic tobacco plants.
Alteration in Rubisco
Outcome
Citation
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
Pale-green plants
Improved zinc accumulation
Pale-green plants
Pale-green plants requiring elevated CO2
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
Yellow plants requiring sucrose
Plants requiring elevated CO2
Plants requiring elevated CO2
Pale-green plants requiring elevated CO2
Plants grow slower than wild type in air
Very low expression of plastome-encoded protein
Insoluble protein
Juvenile plants required elevated CO2
Kanevski and Maliga, 1994
Rumeau et al., 2004
Zhang et al., 2011
Andrews and Whitney, 2003;
Kanevski et al., 1999
Kanevski et al., 1999
Whitney and Andrews, 2001b
Alonso et al., 2009
Whitney et al., 2011b
Whitney et al., 2011b
Whitney and Andrews, 2001a
Whitney et al., 2001
Whitney et al., 2009
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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 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
Page 6 of 12 | Hanson et al.
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 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
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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 CO2 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
Engineering photosynthesis by chloroplast transformation | Page 7 of 12
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)
Fructose-bisphosphate aldolase (FBP aldolase)
ADP-glucose pyrophosphorylase (ADPGPP)
UDP-glucose phosphorylase
Bicarbonate transporters
Rubisco activase
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
Enzymes for reduction of photorespiration
Bacterial glycolate to glycerate pathway (five proteins)
Glycolate catabolic pathway (glycolate oxidase (GO), malate synthase (MS), and catalase (CAT)
Cyanobacterial ictB gene
Kebeish et al., 2007
Fahnenstich et al., 2008; Maier et al., 2012
Lieman-Hurwitz et al., 2003
harmful product. Both bypasses 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.
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
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
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
Page 8 of 12 | Hanson et al.
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.
Bally J, Nadai M, Vitel M, Rolland A, Dumain R, Dubald M.
2009. Plant physiological adaptations to the massive foreign protein
synthesis occurring in recombinant chloroplasts. Plant Physiology 150,
1474–1481.
Acknowledgements
Cardi T, Lenzi P, Maliga P. 2010. Chloroplasts as expression
platforms for plant-produced vaccines. Expert Review of Vaccines 9,
893–911.
References
Ahmad A, Pereira EO, Conley AJ, Richman AS, Menassa R.
2010. Green biofactories: recombinant protein production in plants.
Recent Patents in Biotechnology 4, 242–259.
Ainsworth EA, Ort DR. 2010. How do we improve crop production
in a warming world? Plant Physiology 154, 526–530.
Allison LA, Simon LD, Maliga P. 1996. Deletion of rpoB reveals
a second distinct transcription system in plastids of higher plants.
EMBO Journal 15, 2802–2809.
Alonso H, Blayney MJ, Beck JL, Whitney SM. 2009. Substrateinduced assembly of Methanococcoides burtonii D-ribulose-1,5bisphosphate carboxylase/oxygenase dimers into decamers. Journal
of Biological Chemistry 284, 33876–33882.
Andrews TJ, Whitney SM. 2003. Manipulating ribulose
bisphosphate carboxylase/oxygenase in the chloroplasts of higher
plants. Archives of Biochemistry and Biophysics 414, 159–169.
Apel W, Schulze WX, Bock R. 2010. Identification of protein stability
determinants in chloroplasts. The Plant Journal 63, 636–650.
Barkan A. 2011. Expression of plastid genes: organelle-specific
elaborations on a prokaryotic scaffold. Plant Physiology 155,
1520–1532.
Barkan A, Walker M, Nolasco M, Johnson D. 1994. A nuclear
mutation in maize blocks the processing and translation of several
chloroplast mRNAs and provides evidence for the differential
translation of alternative mRNA forms. EMBO Journal 13, 3170–3181.
Bock R, Khan MS. 2004. Taming plastids for a green future. Trends
in Biotechnology 22, 311–318.
Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM,
Jones AR, Randolph-Anderson BL, Robertson D, Klein TM,
Shark KB 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.
Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z and Maliga
P. 2006. Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts
confers resistance to potato tuber moth. Transgenic Research 15,
481–488.
Corneille S, Lutz K, Svab Z, Maliga P. 2001. Efficient elimination of
selectable marker genes from the plastid genome by the CRE-lox sitespecific recombination system. The Plant Journal 27, 171–178.
Covshoff S, Hibberd JM. 2012. Integrating C4 photosynthesis
into C3 crops to increase yield potential. Current Opinions in
Biotechnology 23, 209–214.
Cui C, Song F, Tan Y, Zhou X, Zhao W, Ma F, Liu Y, Hussain J,
Wang Y, Yang G, He G. 2011. Stable chloroplast transformation of
immature scutella and inflorescences in wheat (Triticum aestivum L.).
Acta Biochimica et Biophysica Sinica 43, 284–291.
Daniell H. 2006. Production of biopharmaceuticals and vaccines
in plants via the chloroplast genome. Biotechnology Journal 1,
1071–1079.
Daniell H, Ruiz G, Denes B, Sandberg L, Langridge W. 2009.
Optimization of codon composition and regulatory elements for
expression of human insulin like growth factor-1 in transgenic
chloroplasts and evaluation of structural identity and function. BMC
Biotechnology 9, 33.
Day A, Goldschmidt-Clermont M. 2011. The chloroplast
transformation toolbox: selectable markers and marker removal. Plant
Biotechnology Journal 9, 540–553.
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.
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
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.
Bally J, Job C, Belghazi M, Job D. 2011. Metabolic adaptation in
transplastomic plants massively accumulating recombinant proteins.
PLoS One 6, e25289.
Engineering photosynthesis by chloroplast transformation | Page 9 of 12
De Marchis F, Pompa A, Bellucci M. 2012. Plastid proteostasis
and heterologous protein accumulation in transplastomic plants. Plant
Physiology 160, 571–581.
Drechsel O, Bock R. 2011. Selection of Shine-Dalgarno sequences
in plastids. Nucleic Acids Research 39, 1427–1438.
Dufourmantel N, Tissot G, Garcon F, Pelissier B, Dubald M.
2006. Stability of soybean recombinant plastome over six generations.
Transgenic Research 15, 305–311.
Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU. 1999. In vivo
analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast
transformation: tobacco plastid gene expression is controlled by
modulation of transcript levels and translation efficiency. The Plant
Journal 19, 333–345.
Fahnenstich H, Scarpeci TE, Valle EM, Flugge UI, Maurino VG.
2008. Generation of hydrogen peroxide in chloroplasts of Arabidopsis
overexpressing glycolate oxidase as an inducible system to study
oxidative stress. Plant Physiology 148, 719–729.
Feng L, Wang K, Li Y, Tan Y, Kong J, Li H, Zhu Y. 2007.
Overexpression of SBPase enhances photosynthesis against high
temperature stress in transgenic rice plants. Plant Cell Reports 26,
1635–1646.
Fukayama H, Ueguchi C, Nishikawa K, Katoh N, Ishikawa C,
Masumoto C, Hatanaka T and Misoo S. 2012. Overexpression of
rubisco activase decreases the photosynthetic CO2 assimilation rate
by reducing rubisco content in rice leaves, Plant and Cell Physiology
53, 976–986.
Golds T, Maliga P, Koop H-U. 1993. Stable plastid transformation in
PEG-treated protoplasts of Nicotiana tabacum. Nature Biotechnology
11, 95–97.
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.
Kajala K, Covshoff S, Karki S, Woodfield H, Tolley BJ, Dionora
MJ, Mogul RT, Mabilangan AE, Danila FR, Hibberd JM, Quick
WP. 2011. Strategies for engineering a two-celled C(4) photosynthetic
pathway into rice. Journal of Experimental Botany 62, 3001–3010.
Kanevski I, Maliga P. 1994. Relocation of the plastid rbcL gene to
the nucleus yields functional ribulose-1,5-bisphosphate carboxylase
in tobacco chloroplasts. Proceedings of the National Academy of
Science USA 91, 1969–1973.
Kanevski I, Maliga P, Rhoades DF, Gutteridge S. 1999. Plastome
engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase in
tobacco to form a sunflower large subunit and tobacco small subunit
hybrid. Plant Physiology 119, 133–142.
Kebeish R, Niessen M, Thiruveedhi K, Bari R, Hirsch HJ,
Rosenkranz R, Stabler N, Schonfeld B, Kreuzaler F, Peterhansel
C. 2007. Chloroplastic photorespiratory bypass increases
photosynthesis and biomass production in Arabidopsis thaliana.
Nature Biotechnology 25, 593–599.
Khan MS. 2007. Engineering photorespiration in chloroplasts: a novel
strategy for increasing biomass production. Trends in Biotechnology
25, 437–440.
Krech K, Ruf S, Masduki FF, Thiele W, Bednarczyk D, Albus
CA, Tiller N, Hasse C, Schottler MA, Bock R. 2012. The plastid
genome-encoded YCF4 protein functions as a nonessential assembly
factor for photosystem I in higher plants. Plant Physiology 159,
579–591.
Kurek I, Chang TK, Bertain SM, Madrigal A, Liu L, Lassner MW,
Zhu G. 2007. Enhanced Thermostability of Arabidopsis Rubisco
activase improves photosynthesis and growth rates under moderate
heat stress. The Plant Cell 19, 3230–3241.
Kuroda H, Maliga P. 2001a. Complementarity of the 16S rRNA
penultimate stem with sequences downstream of the AUG
destabilizes the plastid mRNAs. Nucleic Acids Research 29, 970–975.
Kuroda H, Maliga P. 2001b. Sequences downstream of the
translation initiation codon are important determinants of translation
efficiency in chloroplasts. Plant Physiology 125, 430–436.
Gray BN, Yang H, Ahner BA, Hanson MR. 2011. An efficient
downstream box fusion allows high-level accumulation of active
bacterial beta-glucosidase in tobacco chloroplasts. Plant Molecular
Biology 76, 345–355.
Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong
JJ, Daniell H and 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.
Gruissem W, Barkan A, Deng XW, Stern D. 1988. Transcriptional
and post-transcriptional control of plastid mRNA levels in higher
plants. Trends in Genetics 4, 258–263.
Lefebvre S, Lawson T, Zakhleniuk OV, Lloyd JC, Raines CA,
Fryer M. 2005. Increased sedoheptulose-1,7-bisphosphatase
activity in transgenic tobacco plants stimulates photosynthesis and
growth from an early stage in development. Plant Physiology 138,
451–460.
Hajdukiewicz PT, Allison LA, Maliga P. 1997. The two RNA
polymerases encoded by the nuclear and the plastid compartments
transcribe distinct groups of genes in tobacco plastids. EMBO Journal
16, 4041–4048.
Herz S, Fussl M, Steiger S and Koop HU. 2005. Development of
novel types of plastid transformation vectors and evaluation of factors
controlling expression. Transgenic Research 14, 969–982.
Horvath EM, Peter SO, Joet T, Rumeau D, Cournac L, Horvath
GV, Kavanagh TA, Schafer C, Peltier G, Medgyesy P. 2000.
Targeted inactivation of the plastid ndhB gene in tobacco results in an
Lieman-Hurwitz J, Rachmilevitch S, Mittler R, Marcus Y and
Kaplan A. 2003. Enhanced photosynthesis and growth of transgenic
plants that express ictB, a gene involved in HCO3- accumulation in
cyanobacteria. Plant Biotechnology Journal 1, 43–50.
Lentz EM, Segretin ME, Morgenfeld MM, Wirth SA, Dus Santos
MJ, Mozgovoj MV, Wigdorovitz A, Bravo-Almonacid FF. 2010.
High expression level of a foot and mouth disease virus epitope in
tobacco transplastomic plants. Planta 231, 387–395.
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
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.
enhanced sensitivity of photosynthesis to moderate stomatal closure.
Plant Physiology 123, 1337–1350.
Page 10 of 12 | Hanson et al.
Long SP, Zhu XG, Naidu SL, Ort DR. 2006. Can improvement in
photosynthesis increase crop yields? Plant, Cell & Environment 29,
315–330.
Parry MA, Andralojc PJ, Mitchell RA, Madgwick PJ, Keys AJ.
2003. Manipulation of Rubisco: the amount, activity, function and
regulation. Journal of Experimental Botany 54, 1321–1333.
Lössl A, Eibl C, Harloff HJ, Jung C, Koop HU. 2003. Polyester
synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant
contents of polyhydroxybutyrate are associated with growth reduction.
Plant Cell Reports 21, 891–899.
Parry MA, Reynolds M, Salvucci ME, Raines C, Andralojc
PJ, Zhu XG, Price GD, Condon AG, Furbank RT. 2011. Raising
yield potential of wheat. II. Increasing photosynthetic capacity and
efficiency. Journal of Experimental Botany 62, 453–467.
Lössl A, Bohmert K, Harloff H, Eibl C, Muhlbauer 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.
Peterhansel C. 2011. Best practice procedures for the establishment
of a C(4) cycle in transgenic C(3) plants. Journal of Experimental
Botany 62, 3011–3019.
Lutz KA, Bosacchi MH, Maliga P. 2006. Plastid marker-gene
excision by transiently expressed CRE recombinase. The Plant Journal
45, 447–456.
Maier A, Fahnenstich H, von Caemmerer S, Engqvist MK,
Weber AP, Flugge UI, Maurino VG. 2012. Transgenic introduction of
a glycolate oxidative cycle into A. thaliana chloroplasts leads to growth
improvement. Frontiers in Plant Science 3, 38.
Maliga P. 2004. Plastid transformation in higher plants. Annual
Reviews of Plant Biology 55, 289–313.
Maliga P, Bock R. 2011. Plastid biotechnology: food, fuel, and
medicine for the 21st century. Plant Physiology 155, 1501–1510.
Mallory AC, Parks G, Endres MW, Baulcombe D, Bowman LH,
Pruss GJ, Vance VB. 2002. The amplicon-plus system for highlevel expression of transgenes in plants. Nature Biotechnology 20,
622–625.
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.
Biotechnology (NY) 13, 362–365.
Monde RA, Greene JC, Stern DB. 2000a. The sequence and
secondary structure of the 3’-UTR affect 3’-end maturation, RNA
accumulation, and translation in tobacco chloroplasts. Plant Molecular
Biology 44, 529–542.
Monde RA, Schuster G, Stern DB. 2000b. Processing and
degradation of chloroplast mRNA. Biochimie 82, 573–582.
Mueller-Cajar O, Whitney SM. 2008. Directing the evolution of
Rubisco and Rubisco activase: first impressions of a new tool for
photosynthesis research. Photosynthesis Research 98, 667–675.
Peterhansel C, Niessen M, Kebeish RM. 2008. Metabolic
engineering towards the enhancement of photosynthesis.
Photochemistry and Photobiology 84, 1317–1323.
Price GD, Badger MR, von Caemmerer S. 2011. The prospect
of using cyanobacterial bicarbonate transporters to improve leaf
photosynthesis in C3 crop plants. Plant Physiology 155, 20–26.
Raines CA. 2006. Transgenic approaches to manipulate the
environmental responses of the C3 carbon fixation cycle. Plant, Cell &
Environment 29, 331–339.
Raines CA. 2011. Increasing photosynthetic carbon assimilation in
C3 plants to improve crop yield: current and future strategies. Plant
Physiology 155, 36–42.
Rosenthal DM, Locke AM, Khozaei M, Raines CA, Long SP, Ort
DR. 2011. Over-expressing the C(3) photosynthesis cycle enzyme
Sedoheptulose-1–7 Bisphosphatase improves photosynthetic carbon
gain and yield under fully open air CO(2) fumigation (FACE). BMC Plant
Biology 11, 123.
Ruan CJ, Shao HB, Teixeira da Silva JA. 2012. A critical review on
the improvement of photosynthetic carbon assimilation in C3 plants
using genetic engineering. Critical Reviews in Biotechnology 32,
1–21.
Ruf S, Hermann M, Berger IJ, Carrer H and Bock R. 2001. Stable
genetic transformation of tomato plastids and expression of a foreign
protein in fruit, Nature Biotechnology 19, 870–875.
Ruf S, Karcher D, Bock R. 2007. Determining the transgene
containment level provided by chloroplast transformation. Proceedings
of the National Academy of Science USA 104, 6998–7002.
Ruhlman T, Verma D, Samson N, Daniell H. 2010. The role of
heterologous chloroplast sequence elements in transgene integration
and expression. Plant Physiology 152, 2088–2104.
Muhlbauer SK, Koop HU. 2005. External control of transgene
expression in tobacco plastids using the bacterial lac repressor. The
Plant Journal 43, 941–946.
Rumeau D, Becuwe-Linka N, Beyly A, Carrier P, Cuine S, Genty
B, Medgyesy P, Horvath E, Peltier G. 2004. Increased zinc content
in transplastomic tobacco plants expressing a polyhistidine-tagged
Rubisco large subunit. Plant Biotechnology Journal 2, 389–399.
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.
Schottler MA, Albus CA, Bock R. 2011. Photosystem I: its
biogenesis and function in higher plants. Journal of Plant Physiology
168, 1452–1461.
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 Science USA 106,
6579–6584.
Scotti N, Rigano MM, Cardi T. 2012. Production of foreign proteins
using plastid transformation. Biotechnology Advances 30, 387–397.
Sharwood RE, von Caemmerer S, Maliga P, Whitney SM. 2008.
The catalytic properties of hybrid Rubisco comprising tobacco small
and sunflower large subunits mirror the kinetically equivalent source
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
Magee AM, MacLean D, Gray JC, Kavanagh TA. 2007. Disruption
of essential plastid gene expression caused by T7 RNA polymerasemediated transcription of plastid transgenes during early seedling
development. Transgenic Research 16, 415–428.
Peterhansel C, Maurino VG. 2011. Photorespiration redesigned.
Plant Physiology 155, 49–55.
Engineering photosynthesis by chloroplast transformation | Page 11 of 12
Rubiscos and can support tobacco growth. Plant Physiology 146,
83–96.
Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub
JM and 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.
Whitney SM, Andrews TJ. 2001a. The gene for the ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit
relocated to the plastid genome of tobacco directs the synthesis
of small subunits that assemble into Rubisco. The Plant Cell 13,
193–205.
Sprengart ML, Fuchs E, Porter AG. 1996. The downstream box: an
efficient and independent translation initiation signal in Escherichia coli.
EMBO Journal 15, 665–674.
Whitney SM, Andrews TJ. 2003. Photosynthesis and growth of
tobacco with a substituted bacterial Rubisco mirror the properties of
the introduced enzyme. Plant Physiology 133, 287–294.
Staub JM, Maliga P. 1995. Expression of a chimeric uidA gene
indicates that polycistronic mRNAs are efficiently translated in tobacco
plastids. The Plant Journal 7, 845–848.
Whitney SM, Sharwood RE. 2008. Construction of a tobacco
master line to improve Rubisco engineering in chloroplasts. Journal of
Experimental Botany 59, 1909–1921.
Stern DB and Gruissem W. 1987. Control of plastid gene
expression: 3’ inverted repeats act as mRNA processing and
stabilizing elements, but do not terminate transcription. Cell 51,
1145–1157.
Whitney SM, Baldet P, Hudson GS, Andrews TJ. 2001. Form
I Rubiscos from non-green algae are expressed abundantly but
not assembled in tobacco chloroplasts. The Plant Journal 26,
535–547.
Stern DB, Goldschmidt-Clermont M, Hanson MR. 2010.
Chloroplast RNA metabolism. Annual Reviews of Plant Biology 61,
125–155.
Whitney SM, Kane HJ, Houtz RL, Sharwood RE. 2009. Rubisco
oligomers composed of linked small and large subunits assemble
in tobacco plastids and have higher affinities for CO2 and O2. Plant
Physiology 149, 1887–1895.
Suzuki Y, Ohkubo M, Hatakeyama H, Ohashi K, Yoshizawa
R, Kojima S, Hayakawa T, Yamaya T, Mae T, Makino A. 2007.
Increased Rubisco content in transgenic rice transformed with the
‘sense’ rbcS gene. Plant Cell Physiology 48, 626–637.
Svab Z, Maliga P. 1993. High-frequency plastid transformation in
tobacco by selection for a chimeric aadA gene. Proceedings of the
National Academy of Science USA 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
Science USA 104, 7003–7008.
Theobald JC, Mitchell RA, Parry MA, Lawlor DW. 1998.
Estimating the excess investment in ribulose-1,5-bisphosphate
carboxylase/oxygenase in leaves of spring wheat grown under
elevated CO2. Plant Physiology 118, 945–955.
Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F,
Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga
P. 2003. Expression of tetanus toxin Fragment C in tobacco
chloroplasts. Nucleic Acids Research 31, 1174–1179.
Uematsu K, Suzuki N, Iwamae T, Inui M and Yukawa H. 2012.
Increased fructose 1,6-bisphosphate aldolase in plastids enhances
growth and photosynthesis of tobacco plants. Journal of Experimental
Botany 63, 3001–3009.
Verhounig A, Karcher D, Bock R. 2010. Inducible gene expression
from the plastid genome by a synthetic riboswitch. Proceedings of the
National Academy of Science USA 107, 6204–6209.
von Caemmerer S, Evans JR. 2010. Enhancing C3 photosynthesis.
Plant Physiology 154, 589–592.
von Caemmerer S, Evans JR, Hudson GS, Andrews TJ. 1994.
The kinetics of ribulose-l,5-bisphosphate carboxylase/oxygenase
in vivo inferred from measurements of photosynthesis in leavesof
transgenic tobacco. Planta 195, 85–97.
Whitney SM, Houtz RL, Alonso H. 2011a. Advancing our
understanding and capacity to engineer nature’s CO2-sequestering
enzyme, Rubisco. Plant Physiology 155, 27–35.
Whitney SM, Sharwood RE, Orr D, White SJ, Alonso H,
Galmes J. 2011b. Isoleucine 309 acts as a C4 catalytic switch that
increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco)
carboxylation rate in Flaveria. Proceedings of the National Academy of
Science USA 108, 14688–14693.
Yabuta Y, Tamoi M, Yamamoto K, Tomizawa K, Yokota A,
Shigeoka S. 2008. Molecular design of photosynthesis-elevated
chloroplasts for mass accumulation of a foreign protein. Plant and Cell
Physiology 49, 375–385.
Yang H, Gray BN, Ahner BA, Hanson MR. 2012. Bacteriophage
5’ untranslated regions for control of plastid transgene expression.
Planta, in press.
Ye GN, Hajdukiewicz PT, Broyles D, Rodriguez D, Xu CW, Nehra
N and Staub JM. 2001. Plastid-expressed 5-enolpyruvylshikimate3-phosphate synthase genes provide high level glyphosate tolerance
in tobacco. Plant Journal 25, 261–270.
Yin Z, Plader W, Malepszy S. 2004. Transgene inheritance in plants.
Journal of Applied Genetics 45, 127–144.
Zhang XH, Webb J, Huang YH, Lin L, Tang RS, Liu A. 2011.
Hybrid Rubisco of tomato large subunits and tobacco small
subunits is functional in tobacco plants. Plant Science 180,
480–488.
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.
Zhu XG, de Sturler E, Long SP. 2007. Optimizing the distribution of
resources between enzymes of carbon metabolism can dramatically
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013
Singh AK, Verma SS, Bansal KC. 2010. Plastid transformation
in eggplant (Solanum melongena L.). Transgenic Research 19,
113–119.
Whitney SM, Andrews TJ. 2001b. Plastome-encoded bacterial
ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)
supports photosynthesis and growth in tobacco. Proceedings of the
National Academy of Science USA 98, 14738–14743.
Page 12 of 12 | Hanson et al.
increase photosynthetic rate: a numerical simulation using an
evolutionary algorithm. Plant Physiology 145, 513–526.
Zhu XG, Long SP, Ort DR. 2010a. Improving photosynthetic
efficiency for greater yield. Annual Reviews of Plant Biology 61,
235–261.
Zhu XG, Shan L, Wang Y, Quick WP. 2010b. C4 rice - an ideal
arena for systems biology research. Journal of Integrated Plant
Biology 52, 762–770.
Zubkot MK, Zubkot EI, van Zuilen K, Meyer P, Day A. 2004. Stable
transformation of petunia plastids. Transgenic Research 13, 523–530.
Downloaded from http://jxb.oxfordjournals.org/ at University of Wisconsin-Madison on February 11, 2013