164
Engineering the plastid genome of higher plants
Pal Maliga
The plastid genome of higher plants is an attractive target for
engineering because it provides readily obtainable high protein
levels, the feasibility of expressing multiple proteins from
polycistronic mRNAs and gene containment through the lack of
pollen transmission. A chloroplast-based expression system that
is suitable for the commercial production of recombinant
proteins in tobacco leaves has been developed recently. This
expression system includes vectors, expression cassettes and
site-specific recombinases for the selective elimination of
marker genes. Progress in expressing proteins that are
biomedically relevant, in engineering metabolic pathways, and in
manipulating photosynthesis and agronomic traits is discussed,
as are the problems of implementing the technology in crops.
Addresses
Waksman Institute, Rutgers, The State University of New Jersey,
190 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, USA;
e-mail: [email protected]
Current Opinion in Plant Biology 2002, 5:164–172
1369-5266/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 8 February 2002
Abbreviations
AAD
aminoglycoside 3′-adenyltransferase
EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
GFP
green fluorescent protein
MCS
multiple cloning site
NPTII
neomycin phosphotransferase II
PAT
phosphinothricin acetyltransferase
PEP
plastid-encoded plastid RNA polymerase
pPRV
plastid repeat vector
PPT
phosphinothricin
Prrn
rRNA operon promoter
RBS
ribosome binding site
5′′-UTR 5′-untranslated mRNA region
T7g10 T7-phage gene 10
TSP
total soluble cellular protein
Introduction
Plastids of higher plants are cellular organelles with circular,
double-stranded genomes of 120–160 kilobases in size.
The genome of each plastid encodes approximately
120 genes: each cell contains up to 10 000 identical copies
of each plastid gene. To obtain a genetically stable plant
all genome copies have to be uniformly transformed.
Plastid transformation is obtained through the following
steps: first, the introduction of transforming DNA that
encodes a selectable marker (e.g. an antibiotic resistance
gene) by the biolistic process or by polyethylene glycol
treatment; second, the integration of the transforming
DNA by two homologous recombination events (Figure 1);
and third, the gradual elimination of wildtype genome copies
during repeated cell divisions on a selective medium [1•].
This review focuses on the production of recombinant
proteins in the chloroplasts of higher plants. Most of the
examples discussed utilize tobacco, as this is the only
species in which plastid transformation is routinely
obtained. Emerging applications for plastid transformation,
progress in extending the technology to new crops and
the potential for gene containment using plastid transformation are also discussed. Other applications of plastid
transformation in higher plants and in Chlamydomonas, a
unicellular alga, have been reviewed elsewhere [1•,2,3].
Vectors
Plastid transformation vectors utilize spectinomycin and
streptomycin resistance as selective markers conferred by
the bacterial aadA gene, which encodes aminoglycoside
3′-adenyltransferase (AAD). The plastid repeat vector
(pPRV) series [4] and the vectors pRB94 and pRB95 [5••]
are advanced vectors, in which a chimeric aadA is linked to
unique restriction sites known as multiple cloning sites
(MCS) (Figure 2). The pPRV vectors target insertions in
the trnV–rps12/7 intergenic region, whereas pRB94 and
pRB95 target the trnfM–trnG intergenic region. The
shuffling of expression signals and coding regions via
unique restriction sites in the MCS facilitates the modular
construction of chimeric genes (Figure 3).
Read-through transcription facilitates the expression of
ribosome binding site (RBS)-coding region segments that are
inserted at intergenic regions, allowing the protein of interest
to be translated from a polycistronic mRNA. This approach
has been adopted for the expression of heterologous proteins
by taking advantage of unique XbaI and SpeI restriction
sites downstream of aadA in the plasmid pZS197 [6]. The
RBS-coding region segments were cloned either directly in
plasmid pZS197 [7,8] or after inserting aadA in the trnI–trnA
intergenic region of a universal vector [9••,10,11]. Readthrough transcription is undesirable if expression of the plastid
transgene is regulated transcriptionally [2]. For information
on read-through transcription in vectors see Figure 2.
Marker genes
The choice of selective marker gene and selective agent
are critical for successful transformation. Selective plastid
markers are spectinomycin–streptomycin and kanamycin
resistance, conferred by the expression of chimeric aadA [6]
and neo genes [12], respectively. A new positive-selection
scheme involves the identification of transplastomic tobacco
lines by their resistance to betaine aldehyde conferred by
expression of betaine aldehyde dehydrogenase, which is
encoded by a plant nuclear gene [13•]. The bacterial bar
gene, encoding phosphinothricin acetyltransferase (PAT),
has also been tested as a plastid marker. When expressed
in the nucleus, PAT confers resistance to the herbicide
phosphinothricin (PPT) and is an excellent marker for
the selection of nuclear gene transformants. Expression of
the bar gene in plastids confers PPT resistance when
Engineering the plastid genome of higher plants Maliga
165
Figure 1
A transformed plastid genome is formed by
two recombination events that are targeted by
homologous sequences. The plastid genome
segments that are included in the vector are
marked as the left (LTR) and right targeting
regions (RTR).
Vector
LTR
LTR
Marker
gene
Gene of
interest
RTR
Wildtype
plastid LTR
genome RTR
Marker
gene
Transformed
plastid
genome
Gene of
interest
RTR
Current Opinion in Plant Biology
introduced by selection for a linked aadA (spectinomycin
resistance) gene. However, bar was not found to be suitable
for the direct selection of transplastomic lines, even when
expressed at a high level (when PAT may accumulate to
7% of total soluble cellular protein [TSP]). Thus, it appears
that the subcellular localization of the gene encoding the
detoxifying enzyme PAT is crucial when directly selecting
by herbicide resistance [14••].
A negative-selection scheme has been developed that is
based on the expression of the bacterial enzyme cytosine
deaminase in plastids [15]. Cytosine deaminase catalyzes
the deamination of cytosine to uracil. 5-fluorocytosine is
toxic to cells that express cytosine deaminase because this
enzyme converts 5-fluorocytosine to the toxic 5-fluorouracil.
This negative selection scheme was utilized to identify
seedlings on 5-fluorocytosine-medium from which
codA was removed by the P1 bacteriophage site-specific
recombinase CRE-lox [16••].
β-glucuronidase (GUS) and, more recently, green fluorescent
protein (GFP) have been used as plastid reporter enzymes.
The enzymatic activity of GUS can be visualized by
histochemical staining [17], whereas GFP is a visual
marker that allows the direct imaging of the fluorescent
gene product in living cells. The GFP chromophore forms
autocatalytically in the presence of oxygen and fluoresces
green when absorbing blue or ultra-violet light. GFP has
been used to detect transient gene expression [18] and
stable transformation events [19,20] in chloroplasts. GFP
Figure 2
MCS
(a)
(RI)
pPRV 111/112
(B)
Sp
aadA
aadA
rrn16
trnV
rps12/7
ORF70B
(b)
(RI)
pSBL-CTV2
X Sp
rrn16
H
aadA
trnA
trnI
MCS
(c)
?
?
(S)
psaB
P
trnS
ycf9
trnG
aadA
trnfM
pRB94/95
rps14
Plastid transformation vectors. (a) The pPRV
vector family target insertions in the
trnV–rps12/7 intergenic region [4]. There is no
read-through transcription of transgenes from
the rrn operon. In the pPRV111 and pPRV112
series, aadA is expressed from alternative
signals and the pUC vector MCS is available in
both orientations. (b) Universal plastid vector
[9••,10,11] targets insertions in the trnI and
trnA intergenic region in the rrn operon. The
insertion targets unique XbaI (X) and SpeI (Sp)
sites in TpsbA derived from plasmid pZS197
[6]. The aadA gene is transcribed from two
promoters: one directly upstream of aadA and
a second upstream of the rrn operon. (c) The
pRB94/pRB95 vectors target the trnf–trnG
intergenic region. Vectors differ with respect to
the orientation of the Bluescript plasmid MCS.
No information has yet been published on readthrough transcription at the MCS. Red,
horizontal wavy lines represent transcripts; their
thickness indicates the relative abundance of
their mRNA. The plastid genes marked are:
ORF70B, psaB, psbD, rrn16, trnV, rps12/7,
rps14, trnA, trnfM, trnG, trnI, trnS and ycf9.
Restriction enzyme recognition sites are
labeled BglII (B), EcoRI(RI), HindII (H), PstI (P),
SpeI (Sp), StuI (S) and XbaI (X). Restriction
sites removed during construction are in
brackets. PL cassettes are symbolized with
boxes shaded with horizontal lines. T cassettes
are shaded with vertical lines.
psbD
Current Opinion in Plant Biology
166
Plant biotechnology
Figure 3
Stem-loop
AUG
UGA
ATG
mRNA
TGA
Xba I
Nco I
Nhe I
Eco RI
Sac I
3′-UTR
PL
T
ATG
PEP
(b)
Coding region
TGA
DNA
PL
Coding region
Xba I
Nhe I
Eco RI
Sac I
–35 –10
Promoter
+ 5′-TCR
Transcription
Hind III
DNA
–35 –10
Promoter
+ 5′-UTR
Translation
3′-UTR
T
Hind III
PEP
(a)
Stem-loop
Ribosome
Modular design of PL and T cassettes with
DNA and RNA signals for transgene
expression. The PL cassette encodes a
promoter and (a) a 5′-UTR and (b) a 5′-TCR
[25••,28••]. The PL cassettes are included in
EcoRI and/or SacI (at the 5′ end) and NcoI
and/or NheI (at the 3′ end) fragments. The
conserved –10 and –35 elements of the rrn
PEP promoter are marked. The coding region
of proteins to be expressed is included in
NheI/XbaI or NcoI/XbaI fragments. ATG and
TGA mark the translation initiation and
termination (stop) codons. T cassettes,
encoding the 3′-UTR, are included in
XbaI–HindIII fragments. Stem-loop structures
formed by nucleotide pairing in the 5′-UTR
and 3′-UTR and in the AUG translation
initiation and UGA stop codons are marked in
the mRNA.
Current Opinion in Plant Biology
has also been fused with AAD and used as a bifunctional
visual and selective marker gene [21].
Expression cassettes
To obtain high levels of protein expression several criteria
must be met. First, the mRNA must accumulate to a high
level as ensured by a strong promoter and stable mRNA.
mRNA stability is determined by the 5′ untranslated
mRNA region (5′-UTR) and the 3′-UTR. Second, the
mRNA must be translated efficiently. The rate-limiting
step of translation is its initiation, which is facilitated by
the 5′-UTR. The 5′-UTR is involved in mRNA–rRNA
interactions (between the mRNA ribosome-binding site
and 16S rRNA 3′-end) and interactions with translationalactivating proteins that facilitate loading onto ribosomes.
Third, the protein should be stable.
Plastid expression cassettes consist of a 5′-regulatory
region (PL cassette) and a 3′-regulatory region (T cassette)
and have convenient restriction sites at their boundaries to
facilitate cloning (Figure 3). The PL cassette includes a
promoter and a transcribed region to control translation.
The translation control sequences may be the mRNA
5′-UTR (Figure 3a) or the 5′ translation control region
(5′-TCR), which includes the 5′-UTR and the aminoterminus of the coding region (Figure 3b). Promoters in
the plastid genome are recognized by the multisubunit
plastid-encoded plastid RNA polymerase (PEP) or the
phage-type nucleus-encoded plastid RNA polymerases
(NEP) [22•]. Most studies have focused on the strong
sigma70-type PEP promoter of the rRNA operon promoter
(Prrn). Prrn can be fused with translation control sequences
of plastid and phage origin to facilitate the translation of
recombinant proteins. The 5′-UTR of Prrn derivatives
listed in Table 1 are truncated and mutant forms of native
plastid or phage translation control sequences.
The T cassette encodes the mRNA 3′-UTR, typically
including a stem-loop structure (Figure 3). The 3′-regulatory
region is important for mRNA stability and functions as
an inefficient terminator of transcription [23]. Most
commonly used T cassettes derive from the plastid psbA,
rbcL and rps16 genes (Table 1). The psbA and rbcL 3′-UTRs
appear to yield more stable mRNAs than the rps16 3′-UTR.
Manipulation of protein levels by engineering
the mRNA 5′-UTR and coding region
amino-terminus
Expression levels from Prrn derivatives vary from low
protein levels (0.001% of TSP) up to 45% of TSP, underlining the importance of posttranscriptional regulation in
plastid gene expression. Protein yield depends on the
mRNA sequence, which gives rise to secondary structures
and interacts with the 16S rRNA 3′-end and translationactivating proteins [24]. Specific PLrrn cassettes tend to
yield consistently high (or low) protein levels when
expressing different proteins (Table 1). The inclusion of
the amino-terminus of the coding region of the source
plastid gene tends to enhance protein accumulation (by
approximately two-fold), probably because the sequences
around the AUG translation initiation codon have evolved
together to ensure the translation of the encoded protein.
For example, neomycin phosphotransferase II (NPTII)
comprised 10.8% and 4.7% of TSP when expressed from a
cassette including the rbcL (pHK34/pHK35) construct pair
with and without a coding region amino-terminal segment,
respectively. For the atpB (pHK30/pHK31) construct pair,
these values were 7.0% and 2.5%, respectively. Silent
mutations in the amino-terminus of the coding region
reduced translation efficiency causing a 35-fold reduction in
the NPTII levels (from 10.8% of TSP to 0.31%) [25••]. The
opportunity to improve protein expression by manipulating
the amino-terminus of the coding-region has been
Engineering the plastid genome of higher plants Maliga
167
Table 1.
Examples of high-level accumulation of recombinant proteins in chloroplasts expressed from plastid transgenes with Prrn
promoter derivatives*.
Protein
TSP
Plasmid
concentration
Promoter 5-UTR†
Amino-terminal
fusion
Coding region
3-UTR
NPTII
NPTII
NPTII
NPTII
NPTII
NPTII
NPTII
1.0%
2.5%
7.0%
4.7%
10.8%
23.0%
16.5%
pTNH32
pHK31
pNK30
pHK35
pHK34
pHK40
pHK38
Prrn
Prrn
Prrn(1)
Prrn
Prrn(2)
Prrn
Prrn
rbcL
atpB
atpB
rbcL
rbcL
T7g10
T7g10
rbcL; 5 AA
–
atpB; 14 AA
–
rbcL; 14 AA
–
T7g10;14 AA
neo
neo
neo
neo
neo
neo
neo
psbA
rbcL
rbcL
rbcL
rbcL
rbcL
rbcL
[12]
[48]
[25]
[48]
[25]
[28]
[28]
EPSPS
EPSPS
0.3%
>10.0%
pMON38798
pMON45259
Prrn
Prrn
T7g10
T7g10
–
GFP; 14 AA
CP4
CP4
rps16
rps16
[26]
[26]
GFP
GFP
5.0%
5.0%
pMON30125
MR220
Prrn(3)
Prrn(3)
rbcL
rbcL
–
–
gfp
gfp
rps16
rps16
[19]
[20]
AAD–GFP
AAD–GFP
8.0%
18.0%
pMSK56
pMSK57
Prrn(1)
Prrn(2)
atpB
rbcL
atpB; 14 AA
rbcL; 14 AA
AadA–gfp
AadA–gfp
psbA
psbA
[21]
[21]
Ubiquitin–
somatotropin
7.0%
pMON38794
Prrn
T7g10
–
rps16
[27]
PAT
PAT
>7.0%
>7.0%
pKO3
pKO18
Prrn(1)
Prrn(1)
atpB
atpB
atpB; 14 AA
atpB; 14 AA
s-bar
b-bar2
rbcL
rbcL
[14]
[14]
Cry1Ac
Cry2Aa2
Cry2Aa2,
20 kDa;
29 kDa;
CTB
5.0%
3.0%
45.3%
pZS224
Prrn(3)
pZS–KM–cry2A
pLD–BD Cry2Aa2 2x-Prrn
rbcL
Bt
Bt
–
–
–
cry1A(c)
cry2Aa2
orf1–orf2—cry2Aa2
rps16
?
psbA
[38]
[8]
[9]
4.1%
pLD-LH-CTB
Reference
2x-Prrn
Synthetic –
ctxB
psbA
[11]
GGAGG
*Identical Prrn derivatives (PL cassettes) in different constructs are identified by arbitrary number. Prrn derivatives that are not numbered are
unique in some detail of the PL cassette. †Same origin of 5-UTR does not mean identical 5-UTR sequences. For example, plasmids pTNH32
and pHK35 are listed to encode a neo gene with a rbcL 5-UTR. However, the actual neo mRNA5-UTR encoded in plasmid pTNH32 includes
18 nucleotides of rbcL 5 UTR fused with 34 nucleotides of the rrn transcript, whereas the pHK35-encoded neo 5-UTR includes 58
nucleotides of the 182-nt native rbcL 5-UTR. AA, amino acids.
demonstrated by a more than 30-fold increase in CP4
ESPS accumulation after fusion with 14 amino-terminal
amino acids of GFP [26••]. Fusion of the human somatotropin
with ubiquitin [27••] and GFP with AAD [21] increased
levels of protein accumulation. This may be due to enhanced
translation rates, enhanced protein stability or both.
Protein accumulation from heterologous translation control
signals incorporated with the heterologous coding region
depends on their recognition in chloroplasts. Translation
signals controlling the Bacillus thuringiensis cry2Aa2 operon
were efficiently recognized, yielding 45.3% TSP as
Cry2Aa2 protein crystals [9••]. Translation control signals
may only be part of the reason for this high-level expression
as the co-expression of two helper proteins alongside the
protoxin facilitated the formation of stable protein crystals.
Levels of expression of the Cry2Aa2 protein alone (without
the helper protein, 2–3% of TSP) [8] and cholera toxin B
subunit (4.1% of TSP) [11] were satisfactory, but not
unusually high. The low level of a protein-based polymer
from a different RBS-coding region segment, despite the
high level of mRNA encoding it, is a reminder that it may
be risky to include untested translation control elements
from a heterologous source [10].
Although the plastid expression system is, in general,
prokaryotic, the rules of prokaryotic gene expression do
not always apply to it. Increasing complementarity
between sequences downstream of the T7-phage gene 10
(T7g10) AUG and the penultimate stem of the 16S rRNA
3′-region reduced NPTII accumulation 100-fold by
reducing translation efficiency and destabilizing the
NPTII mRNA [28••]. In E. coli, similar changes would
enhance rather than reduce protein accumulation.
Expression of heterologous proteins: codon
usage and mRNA stability
The expression of proteins encoded by eukaryotic nuclear
genes in E. coli is often problematic because of differences
in codon usage and/or degradation of the eukaryotic
mRNA. In tobacco plastids, with the exception of the
borderline CGC codon (which appears 3.9 times per
1000 codons), all codons are used more frequently than
4.3 times per 1000 codons, a frequency below which
problems were encountered when expressing heterologous
proteins in E. coli. Indeed, codon optimization in plastids
has so far yielded at best a few-fold increase in protein
yield. Protein accumulation from a synthetic, codonoptimized version of CP4 (containing 77% plastid-preferred
168
Plant biotechnology
Figure 4
Observed gene deletions
(a)
bar
aadA
aadA + bar
uidA + aadA
rbcL P1 uidA
Transplastome (Tpt)
rbcL
aadA
T1 P1′
P1
bar
T1 P1
T1
bar
T1
Product of desired
deletion via P1 repeat
(b)
S2
S1
N
Wildtype (Wt)
N
N
Wt + Tpt
N
N
Wt + Tpt + bar +
(uidA + bar )
N
uidA + bar
Tpt
bar
uidA + bar
(Rare)
Tpt
(Frequent)
bar
(Rare)
Current Opinion in Plant Biology
Elimination of marker genes by loop-out via short, directly repeated
sequences. (a) Map of the transformed plastid genome with a reporter
gene (uidA), a selective marker (aadA) and a herbicide resistance
gene (bar). P1 promoter fragments (of 174 basepairs) and the
418-basepair TpsbA cassettes (T1) are short, direct repeats. Breaks in
the black, orange and blue bars represent gene deletions detected by
Iamtham and Day [29]. (b) Multiple, alternative recombination events
and segregation of the plastid genome yield marker-free herbicide
resistant plants. Note that selection for spectinomycin resistance (S1)
may be followed by selection for herbicide resistance (S2) that
eliminates the non-herbicide resistant deletion derivatives. N, nucleus.
codons) was just 1.5–2.0-fold higher than from the
bacterial CP4 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS) coding region (which contains 44% plastidpreferred codons) [26••].
Neither codon usage nor mRNA stability was reported to
be a problem when expressing the human somatotropin
cDNA in chloroplasts. Protein levels were reasonably high
(7% of TSP) as long as the somatotropin-coding region
was translationally fused with ubiquitin and expressed
from a modified T7g10 leader [27••]. Data on further
human proteins are required to fully assess the problems
associated with expressing human cDNAs in chloroplasts.
Marker elimination systems
Expression of the selective marker at a high level may be
desired to ensure selective amplification when only a
few transformed genome copies are present. When the
marker gene is present in each plastid genome, however,
the marker protein may make up as much as 10% of TSP,
a significant metabolic burden on the plant. In addition,
given the concern about gene flow and the possible
health hazards of antibiotic resistance genes, it is desirable
to remove the selective marker once it has fulfilled its
useful purpose.
The scheme for the removal of plastid markers developed
by Iamtham and Day [29] exploits the formation of multiple
recombination products in the same plastid (Figure 4).
Plastids in this scheme are transformed with a construct in
which the selective marker is flanked by short direct
repeats. The repeats are two 174-basepair PLrrn promoter
fragments (P1, light arrow) and three 418-basepair TpsbA
sequences (T1, heavy arrow; Figure 4). P1′ and P1 are also
partially homologous. Heteroplastomic clones obtained
after the delivery of DNA, in which different fortuitous
deletion derivatives form by homologous recombination
via the short direct repeats, are identified by spectinomycin
resistance. Derivatives that lack the marker gene but carry
the gene of interest are identified by herbicide resistance
and DNA-gel-blot analyses after plant regeneration. The
difficulty of this approach is that transformation and the
elimination of marker genes occur simultaneously, making
the process laborious and difficult to control.
Transformation and marker gene elimination are separate
processes in the CRE-lox plastid marker elimination
system developed independently by Hajdukiewicz et al.
[30••] and in our laboratory [16••] (Figure 5). CRE is a
phage site-specific recombinase that efficiently excises
any sequence between two directly oriented 34-basepair
lox-sites. According to the CRE-lox scheme, the marker
gene (flanked by two directly-oriented lox sites) and the
gene of interest are introduced into the plastid genome in
the absence of CRE activity. The transformed plastid
genome is stable until CRE activity is provided from a
nuclear-encoded, plastid-targeted CRE. The nuclear
Cre may be introduced by Agrobacterium-mediated
transformation [16••,30••] or by pollination [16••], and is
subsequently removed by segregation in the seed
progeny. Although the desired marker-free plants can be
readily obtained by this system, unpredicted rearrangements
of the plastid genome were detected in a significant
fraction of the Agrobacterium-transformed clones. It
appears that these rearrangements are much less frequent
in plants obtained by pollination.
Engineering the plastid genome of higher plants Maliga
169
Figure 5
Scheme for the elimination of marker genes
using the CRE-lox site specific recombination
system in plastids. In this example, the
transplastome contains the marker gene aadA
flanked by lox sites (arrowheads). The gene of
interest (goi) that was introduced by selection
for the marker gene is shown in blue.
Introduction of cre (a) by Agrobacterium
transformation or (b) by crossing results in
expression of the CRE site-specific
recombinase from the nuclear cre gene. The
CRE is plastid targeted, and will
simultaneously excise the marker gene from all
the plastid genome copies. In the experiment
described in [16••], the gene to keep (i.e. the
goi) was aadA and the gene to remove was
codA, a negative selectable marker. In [30••],
the gene to be eliminated was aadA and the
gene to keep was gfp (encoding GFP).
Presence of cre and the linked kanamycinresistance (neo) gene in the nuclear genome
are indicated by c and n, respectively; their
absence by a ‘+’. T0 refers to transgenic
plants regenerated from tissue culture; T1 and
T2 are the first and second generation
progeny of T0 produced by self pollination.
Transplastome
goi
rrn16
aadA
(b)
(a)
Introduction
of nuclear
cre
Agrobacterium
transformation
cn
++
T0
rps12
trnV
goi
rrn16
To
rps12
trnV
Pollination
++
++
Excision
via lox
site
cn
T
++ 0
rps12
trnV
goi
rrn16
+
+
aadA
aadA
T0
cn cn cn ++
cn ++ ++ ++
T1
T1
Loss of
cre in
progeny
++ cn cn cn
++ cn ++ ++
T2
Current Opinion in Plant Biology
Applications of plastid transformation
Plastid transformation has been utilized in basic science,
biotechnology and agronomy [1•,2,3]. Recently, progress
has been made in engineering Rubisco, the carbon-dioxidefixing enzyme. The long-term objective of Rubisco
engineering is to replace the existing Rubisco with a more
efficient form. Earlier attempts to replace the tobacco
Rubisco with homologs from sunflower and the cyanobacterium Synechococcus PCC 6301 did not result in
functional hybrid Rubisco [31]. When relocated to the
plastid genome, a tobacco RbcS gene was capable of directing
the synthesis of small Rubisco subunits. However, these
subunits were not abundant, suggesting that either the
translation of the mRNA or the access of the product to the
Rubisco assembly pathway was not efficient [32]. Rubisco
translated from inserted rbcLS operons from two non-green
algae were insoluble [33]. The first success in Rubisco
engineering was the replacement of the tobacco rbcL gene,
which encodes the Rubisco large subunit, with the Form II
Rubisco of Rhodospirillum rubrum, a photosynthetic
bacterium. The transplastomic tobacco plants require
carbon dioxide supplementation, consistent with the
kinetic properties of the bacterial Rubisco [34••]. These
transplastomic tobacco plants are the first photosynthetic
higher plants with a foreign Rubisco that are fully
autotrophic and fertile. They represent a milestone on the
road towards achieving the long-term goal of engineering
improved photosynthetic efficiency.
Metabolic engineering through transformation of the
plastid genome has also been reported. To boost tryptophan
production, a feedback-insensitive tobacco anthranilate
synthase alpha-subunit was overexpressed from a tobacco
cDNA in plastids. Free tryptophan was 10-fold more
prevalent in the transplastomic leaves. Although the
transgene was highly expressed, the overall increase in
170
Plant biotechnology
enzymatic activity was only four-fold, suggesting that the
overexpression of both anthranilate synthase subunits is
necessary to fully exploit the benefits of boosting this
pathway [35]. The desire to increase the lipid content of
seeds is the reason for attempts to boost acetyl-CoA
carboxylase activity by introducing a stronger promoter
upstream of accD by plastid transformation [36].
There is interest in using transgenic plants as a source of
recombinant protein therapeutics and vaccines [37].
Production of human secretory protein somatotropin in
tobacco chloroplasts was the first such application of
plastid transformation. Proper protein folding and the
formation of disulfide bonds normally require passage
through the endoplasmic reticulum. However, the chloroplast-produced somatotropin (which comprised 7% of
TSP) was in a biologically active, soluble and disulfidebonded form, which was nearly devoid of complex
post-transcriptional modifications [27••]. The mature form
of the B subunit of the cholera toxin, a candidate vaccine
antigen, has also been expressed in chloroplasts. The
chloroplast-produced B subunit (which comprised 4.1% of
TSP) assembled into oligomers and was biologically active
in a cell-culture assay [11].
The most common transgenic traits in field grown plants
are resistance to insects and herbicides. Both of these traits
are expressed from nuclear genes but plastid versions of
the insecticidal protein genes are also available [8,9••,38].
Although a petunia EPSPS gene has been expressed in
plastids, the resulting level of glyphosate tolerance was
below that required for field applications [7]. Recently,
strong glyphosate resistance was engineered by the overexpression in plastids of tolerant forms of different
prokaryotic (Achromobacter, Agrobacterium and Bacillus)
EPSPS genes. Greater accumulation of plastid-expressed
EPSPS protein than of EPSPS from nuclear genes was
necessary to confer strong resistance [26••]. However,
field-level tolerance to ‘Liberty’, a herbicide containing
PPT as an active ingredient, was obtained by expression of
bar in the plastid genome. In this case, resistance did not
require high-level accumulation of PAT [14••]. Utilization
of the plastid herbicide-resistance genes will have to wait
until plastid transformation is achieved in major crops.
Implementation of plastid engineering
technology in new crops
Plastid transformation in tobacco [6,39] was followed by
plastid transformation in two other solanaceous species,
potato [19] and tomato [5••]. Plastid transformation in
tomato is a significant breakthrough as tomato fruits were
shown to express relatively high levels (approximately 0.5%
of TSP) of the marker gene product. Significant improvement in the expression level of recombinant proteins in
tomato fruit is anticipated thanks to the use of new, more
sophisticated expression tools. This is the first example of
protein expression in an edible plant part, providing
promise of high-level expression of oral vaccines [5••].
Plastid transformation in Arabidopsis, although feasible, is
very inefficient [40], probably due to the inefficient
incorporation of the transforming DNA. In rice, the plastid
genomes of embryogenic cells could be transformed
relatively easily. However, plant regeneration from cultured
rice cells by standard protocols occurs prior to achieving
the homoplastomic state, making it difficult to obtain
genetically stable transplastomic plants [21]. Problems
such as these that are specific to taxonomic groups, once
recognized, can be overcome. Plastid transformation in
Arabidopsis and rice will be followed by implementation of
protocols in the related crops: oilseed rape, vegetable
brassicas, maize, barley and wheat.
Conclusions and future directions
Tools, including vectors, expression cassettes and systems
for the elimination of marker genes, are available for the
expression of recombinant proteins in tobacco chloroplasts.
These will be combined in a new generation of vectors to
provide high-level protein expression and the convenient
elimination of marker genes. The focus of research in
tobacco will shift to issues related to protein modification,
processing and subunit assembly. Tools for regulated plastid
gene expression are yet to be developed. A T7-phage RNA
polymerase expressed in the nucleus from a chemically
inducible promoter, and driving the expression of transgenes
in plastids from a T7 gene 10 promoter, is the first system
to address this problem [2,41].
Advantages of incorporating the transgenes in the plastid
genome are: containment of transgenes due to the lack of
pollen transmission; expression of multiple genes in
operons; high expression levels; possibility of expressing
unmodified bacterial genes and human cDNAs; and lack
of gene silencing and position effects. The disadvantages
are that the proteins are not exported and the difficulty
of the technology required. To fully benefit from the
technology, experience gained from work on tobacco
should be applied to all crops. The discovery of the right
combination of tissue culture procedures and selective
markers will be crucial for the success of plastid engineering.
Will it be necessary to construct vectors for each crop?
Probably not, although specific vectors may be constructed
for each of the larger taxonomic groups. Plastid transformation in potato [19] and tomato [5••] using tobacco
vectors is encouraging in this regard. There is no need for
perfect homology between the recipient genome and
transformation vector as multiple internal exchange events
result in the mosaic-like integration of donor DNA [42].
The advantage of using homologous targeting sequences
is that only one type of transplastome may form. Speciesspecific differences in recognition of DNA and RNA
signals and in capacity to alter essential mRNA editing
sites will determine how widely the vectors can be used.
Nuclear transgene flow via pollen to wild weedy relatives
can be a problem, especially if gene transfer enhances the
fitness of the weedy relative by conferring resistance to
Engineering the plastid genome of higher plants Maliga
herbicides and insect pests [43]. The incorporation of
transgenes into the plastid genome would provide significant advantages, as plastids in the major crops are not
transmitted by pollen. Although the transgenes would not
leave the transgenic crop in pollen, rare hybrids still could
form if the crop were to be pollinated by the wild relatives
or in mixed stands [44]. Relevant in this regard is that, of
the 13 most important agricultural crops, 12 have a sexually
compatible weedy relative with which they form a hybrid
in some geographic area. An alternative source of plastid
genome transfer to weeds could occur occasionally: paternal
transmission of plastids has been detected under extreme
selection pressure in a tissue-culture system in plants with
an alien cytoplasm or in inter-specific crosses, even in
species with a strict maternal inheritance of plastids [45,46].
Knowing the effort required to achieve the homoplastomic
state in plastid transformation experiments [47], even if
transfer occurs, such plastids are unlikely to become
established in the recipient weed. Nevertheless, the
possibility of genome transfer from the new transplastomic
crops to weedy relatives will have to be evaluated.
Acknowledgements
Research in the author’s laboratory was supported by grants from the
National Science Foundation (MCB 96-30763 and MCB 99-05043), and by
the Rockefeller Foundation Rice Biotechnology Program.
References and recommended reading
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