Journal of Experimental Botany, Vol. 54, No. 381, pp. 179±189, January 2003
DOI: 10.1093/jxb/erg026
REVIEW ARTICLE
Molecular evolution and genetic engineering of C4
photosynthetic enzymes
Mitsue Miyao1
Photosynthesis Laboratory, National Institute of Agrobiological Sciences (NIAS), Kannondai, Tsukuba 305-8602,
Japan
Received 27 May 2002; Accepted 5 September 2002
Introduction
The majority of terrestrial plants, including many
important crops such as rice, wheat, soybean, and
potato, are classi®ed as C3 plants that assimilate
atmospheric CO2 directly through the C3 photosynthetic pathway. C4 plants, such as maize and sugarcane, evolved from C3 plants, acquiring the C4
photosynthetic pathway in addition to the C3 pathway
to achieve high photosynthetic performance and high
water- and nitrogen-use ef®ciencies. Consequently,
the transfer of C4 traits to C3 plants is one strategy
being adopted for improving the photosynthetic performance of C3 plants. The recent application of
recombinant DNA technology has made considerable
progress in the molecular engineering of photosynthetic genes in the past ten years. It has deepened
understanding of the evolutionary scenario of the C4
photosynthetic genes. The strategy, based on the
evolutionary scenario, has enabled enzymes involved
in the C4 pathway to be expressed at high levels and
in desired locations in the leaves of C3 plants.
Although overproduction of a single C4 enzyme can
alter the carbon metabolism of C3 plants, it does not
show any positive effects on photosynthesis.
Transgenic C3 plants overproducing multiple
enzymes are now being produced for improving the
photosynthetic performance of C3 plants.
Terrestrial plants are classi®ed into three major photosynthetic types, namely, C3, C4 and Crassulacean acid
metabolism (CAM) plants, according to the mechanism of
their photosynthetic carbon assimilation. About 90% of
terrestrial plant species, which include major crops such as
rice (Oryza sativa), wheat (Triticum aestivum), soybean
(Glycine max), and potato (Solanum tuberosum), are
classi®ed as C3 plants, and they assimilate CO2 directly
through the C3 photosynthetic pathway, also called the
Calvin cycle or the photosynthetic carbon reduction (PCR)
cycle. C4 and CAM plants possess a unique photosynthetic
pathway, in addition to the C3 pathway, which allows them
to adapt to speci®c environments. While C3 plants grow
well in temperate climates, CAM plants such as stonecrops
and cactus adapt to extreme arid conditions, but their
photosynthetic capacity is very low (Black, 1973). By
contrast, C4 plants such as maize (Zea mays) and sugarcane
(Saccharum of®cinarum) adapt to high light, arid and
warm environments and achieve higher photosynthetic
capacity and higher water- and nitrogen-use ef®ciencies
compared with C3 plants (Black, 1973). Both C4 and CAM
plants evolved from ancestral C3 species in response to
changes in environmental conditions that caused a
decrease in CO2 availability. C4 plants evolved in response
to the low atmospheric CO2 concentrations, while the
CAM plants evolved either in response to the selection of
increased water-use ef®ciency or for increased carbon gain
(Ehleringer and Monson, 1993).
Key
words:
C4
photosynthesis,
gene
evolution,
phosphoenolpyruvate carboxylase, transgenic plants.
1
Fax: +81 298 38 7073. E-mail: [email protected]
Abbreviations: AspAT, aspartate aminotransferase; CAM, Crassulacean acid metabolism; Glc6P, glucose-6-phosphate; GUS, b-glucuronidase; NAD-ME,
NAD-malic enzyme; NADP-MDH, NADP-malate dehydrogenase; NADP-ME, NADP-malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC,
phosphoenolpyruvate carboxylase; PEP-CK, phosphoenolpyruvate carboxykinase; PPDK, pyruvate, orthophosphate dikinase; Rubisco, ribulose-1,5bisphosphate carboxylase/oxygenase.
Journal of Experimental Botany, Vol. 54, No. 381, ã Society for Experimental Biology 2003; all rights reserved
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Abstract
180 Miyao
In leaves of C3 plants, all of the photosynthetic reactions
from the capture of solar light energy to assimilation of
carbon into carbohydrates (triosephosphates) proceed in
the chloroplasts of the mesophyll cells (Fig. 1A). The
primary CO2 ®xation step in the C3 pathway is catalysed
by ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco). However, Rubisco also reacts with O2 at its
catalytic site (oxygenase reaction), leading to photorespiration. Photorespiration plays a role in protecting
photosynthesis from photoinhibition (Osmond and Grace,
1995), but it wastes ®xed carbon as released CO2 and
decreases the ef®ciency of photosynthetic CO2 assimilation in C3 plants (Leegood et al., 1995). Under current
atmospheric conditions (0.036% CO2, 21% O2), up to 50%
of the ®xed carbon is lost by photorespiration. C4 plants
have evolved the C4 photosynthetic pathway, a mechanism
to concentrate CO2 at the site of the reaction of Rubisco,
and thereby overcame photorespiration. This CO2-concentrating mechanism, together with modi®cation of leaf
anatomy, enabled C4 plants to achieve high photosynthetic
ef®ciency (Hatch, 1987).
Leaves of C4 plants have two types of photosynthetic
cells, the mesophyll and bundle sheath cells that contain
chloroplasts of different functions. While all the photo-
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Fig. 1. Simpli®ed illustrations of the C3 photosynthetic pathway (A)
and the C4 photosynthetic pathway of the NADP-ME type C4 plants
(B). In the C4 pathway, one molecule of CO2 is pumped up from the
cytosol of the mesophyll cell into the chloroplast of the bundle sheath
cell where Rubisco is present. This process consumes two molecules
of ATP (one consumed by PPDK and the other required for the
conversion of AMP produced by PPDK to ADP), but it does not
consume or produce any other substances. TP, triosephosphate.
synthetic enzymes are con®ned in the mesophyll cells in
C3 plants, they are localized in the mesophyll and/or
bundle sheath cells in C4 plants. The enzymes involved in
the C3 pathway are located in the chloroplasts of the bundle
sheath cells while those involved in the C4 pathway (C4
photosynthetic enzymes) in the mesophyll and/or bundle
sheath cells (Fig. 1B). The C4 pathway consists of three
key steps: (i) the initial ®xation of CO2 in the cytosol of the
mesophyll cells by phosphoenolpyruvate carboxylase
(PEPC) to form a C4 acid, oxaloacetate (OAA), (ii)
decarboxylation of a C4 acid in the bundle sheath cells to
release CO2, and (iii) regeneration of the primary CO2
acceptor phosphoenolpyruvate (PEP) (Hatch, 1987;
Fig. 1B). As a whole, one molecule of CO2 is pumped
up from the cytosol of the mesophyll cells into the vicinity
of Rubisco in the chloroplast of the bundle sheath cells,
consuming two molecules of ATP. The decarboxylation
reaction is catalysed by one or more of the three enzymes,
namely, NADP-malic enzyme (NADP-ME), NAD-malic
enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK), and C4 plants are classi®ed into three
subtypes depending on the major decarboxylation enzyme.
The C4 acid exported from the mesophyll to bundle sheath
cells are also different. Before being exported, OAA is
reduced to malate by NADP-malate dehydrogenase
(NADP-MDH) or transaminated to aspartate by aspartate
aminotransferase (AspAT) in the NADP-ME type and the
NAD-ME and PEP-CK types, respectively. Regeneration
of PEP is catalysed by pyruvate, orthophosphate dikinase
(PPDK) located in the mesophyll cell chloroplasts in all
subtypes, although PEP-CK in the bundle sheath cell
cytosol also participates in this process in the PEP-CK
type. Maize and sugarcane use NADP-ME for the
decarboxylation and these are classi®ed as the NADPME type.
The rate-limiting steps of the C4 pathway can be
assessed from the control coef®cients (Cj) determined for
the individual enzymes. Cj is de®ned as the ratio between
the fractional change in metabolite ¯ux and the fractional
change in enzyme activity, and a Cj of one indicates that
the ¯ux is fully controlled at this step while the value of
zero indicates no control over the ¯ux (ap Rees and Hill,
1994). The Cj values for C4 enzymes can be determined
from the plot of the CO2 assimilation rate against enzyme
activity, which has been altered by mutation and/or
transgenic techniques. The results indicate that the C4
pathway is controlled by reactions of Rubisco, PEPC and
PPDK (reviewed in Matsuoka et al., 2001). Under
saturating illumination and ambient CO2, the Cj values
for Rubisco, PEPC and PPDK were 0.5±0.6, 0.35, and 0.2±
0.4, respectively. By contrast, two other enzymes examined so far appear to exert little or no control: the Cj values
for NADP-MDH of Flaveria bidentis (NADP-ME type)
and for the decarboxylation enzyme of Amaranthus edulis
(NAD-ME type) were almost zero. The activities of the
Evolution and engineering of C4 enzymes 181
rate-limiting enzymes are strictly regulated in the leaves of
C4 plants. The activity of Rubisco is controlled by the
Rubisco activase as it is in C3 plants (Salvucci et al., 1987).
Those of PEPC and PPDK are regulated through reversible
protein phosphorylation by their speci®c regulatory
proteins, being up-regulated in the light (Burnell and
Hatch, 1985; Vidal and Chollet, 1997).
Since the discovery of the C4 pathway, it has been
postulated that the transfer of C4 traits to C3 plants should
improve the photosynthetic performance of C3 plants.
Initially, conventional hybridization between C3 and C4
plants was carried out. This approach was available only in
several plant genera and most C3-C4 hybrids were infertile
(Brown and Bouton, 1993). Another approach that has
been adopted in the last ten years involves the use of
recombinant DNA technology. With this technology,
understanding of the evolution of C4 photosynthetic
genes has been expanded and it is now possible to express
C4 enzymes at high levels and in desired locations in the
leaves of C3 plants. The evolution of C4 genes together
with techniques with which to overproduce C4 enzymes in
the leaves of C3 plants is summarized here. The regulation
and physiological impacts of overproduced C4 enzymes in
transgenic rice plants are also presented. The physiological
impacts of the overproduction in potato, tobacco
(Nicotiana tabacum) and Arabidopsis thaliana as well as
rice have previously been reviewed in detail by HaÈusler
et al. (2002).
Evolution of C4 photosynthetic genes
C4 photosynthetic genes had previously been considered to
be speci®c for C4 plants, since the activities of the
corresponding enzymes are low in C3 plants (Hatch, 1987)
and their kinetic properties are usually different from those
of C4 enzymes (e.g. for PEPC, see Svensson et al., 1997;
Dong et al., 1998). However, recent comparative studies
have revealed that C3 plants have at least two different
types of genes, one encoding enzymes of `housekeeping'
function and the other very similar to the C4 genes of C4
plants, though expression of the latter is very low or even
undetectable in C3 plants. Based on this ®nding, it is
postulated that the C4 genes evolved from a set of preexisting counterpart genes in ancestral C3 plants, with
modi®cations in the expression level in the leaves and
kinetic properties of enzymes (Ku et al., 1996). Hereafter,
the C4 genes in C4 plants and their homologues in C3 plants
are designated C4-speci®c and C4-like genes, respectively.
In addition to C4-speci®c or C4-like genes, both C3 and C4
plants have other homologous genes for the housekeeping
function. These are designated as C3-speci®c genes. The
number of homologous genes and the evolutionary origins
of C4-speci®c genes are different among C4 enzymes and
plant species (Monson, 1999). By contrast, modi®cations
of C4-like genes required for functioning in the C4 pathway
probably share common features in all the C4-speci®c
genes examined so far. In the following, the evolutionary
origin of the maize C4-speci®c PPDK gene is considered as
an example.
Evolution of the maize C4-speci®c PPDK gene
Maize has three different isoforms of PPDK, namely, the
chloroplastic isoform involved in the C4 pathway and two
cytosolic isoforms (Sheen, 1991). The chloroplastic and
one cytosolic isoforms are encoded by a single gene that
has a dual promoter system (Glackin and Grula, 1990;
Sheen, 1991; Fig. 2). This gene (designated Pdk1 hereafter) has two transcription initiation sites and transcription
from these sites is regulated by different promoters located
at their respective 5¢-¯anking regions. Transcription at the
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Fig. 2. Comparison of the rice and maize Pdk1 genes that encode the chloroplastic and cytosolic isoforms of PPDK. Both genes have a dual
promoter system and transcription starts at two different sites indicated by bent arrows, giving rise to transcripts different in size. The larger
transcript encodes the chloroplastic isoform and the smaller one encodes the cytosolic isoform. The coding regions common to the two transcripts
are represented by ®lled boxes, and the 5¢- and 3¢-non-coding regions by open boxes. Hatched boxes in maize exon 1 and rice exon 1¢ represent
regions that encode the transit peptide, and those in maize exon 2 and rice exon 2 represent the coding regions unique to the small transcript. ATG
and TGA indicate the initiation and termination codons, respectively. The gene structures reported previously (Imaizumi et al., 1997) are
modi®ed.
182 Miyao
®rst initiation site produces large transcripts for the
chloroplastic isoform, while that from the second site
produces small transcripts for the cytosolic isoform. The
large transcripts are expressed highly speci®cally in the
mesophyll cells of green leaves at a high level and
expression is induced by light, while the small transcripts
are expressed at a high level in roots but at a low level in
the mesophyll cells (Sheen, 1991). By de®nition, the genes
encoding the chloroplastic and cytosolic isoforms in the
maize Pdk1 are C4-speci®c and C3-speci®c genes, respectively: they have previously been designated C4ppdkZm1
and cyppdkZm1, respectively, by Sheen (1991). The
second cytosolic isoform is encoded by a gene with a
single promoter (previously designated cyppdkZm2 by
Sheen (1991) but Pdk2 hereafter), which shows high
homology to the C3-speci®c gene in Pdk1. This gene is
expressed at a very low level in the mesophyll cells (Sheen,
1991) and thus C3-speci®c. The cytosolic isoform of PPDK
accumulates signi®cantly in kernels (Aoyagi and Bassham,
1984; Aoyagi and Chua, 1988), though it is uncertain
which of the two genes is expressed in this organ.
Rice also has three different isoforms of PPDK, and two
different genes have been identi®ed. One has a dual
promoter system and encodes the chloroplastic and
cytosolic isoforms (Imaizumi et al., 1997) and the other,
with a single promoter, encodes the cytosolic isoform
(Moons et al., 1998). The former gene is highly
homologous to the maize Pdk1 (Imaizumi et al., 1997).
It has 21 exons and the positions of introns are essentially
the same as those in the maize gene, except that the exon 1
and 3 of the maize gene are split into two exons in the rice
gene (Fig. 2). The deduced amino acid sequences are 88%
homologous in the mature protein portion and 56%
homologous in the transit peptide portion. In addition,
this gene is expressed in rice plants essentially in the same
way as the maize Pdk1 does in maize, except for the
expression level of the large transcripts in green leaves: the
large transcripts are expressed speci®cally in photosynthetic organs but at low levels, while the smaller ones in
reproductive organs at high levels and roots at a low level
(Imaizumi et al., 1997). Thus, this gene is a counterpart of
the maize Pdk1. The other gene encoding the cytosolic
isoform has been identi®ed as a cDNA clone from rice
roots (osppdka; Moons et al., 1998), but its genomic clone
has not yet been isolated. Since the 3¢ region of the cDNA
is highly homologous to the exons in the 3¢ region of the
rice Pdk1 (Moons et al., 1998), it seems likely that this
gene is a counterpart of the maize Pdk2.
From the comparison between the maize and rice genes,
the postulated evolutionary origin of PPDK genes is
depicted in Fig. 3. From a single ancestral gene, two genes
encoding a cytosolic isoform were derived. One was an
ancestral Pdk2 gene, which would evolve to become the
Pdk2 genes of C3 and C4 plants. The other was an ancestral
Pdk1 gene with a single promoter. This gene subsequently
evolved to become the Pdk1 gene with a dual promoter
system in an ancestral C3 plant, acquiring a sequence for
the transit peptide, and ®nally evolved to become the Pdk1
gene of a C4 plant by acquiring a mechanism(s) for highlevel expression. The evolutionary scenario for the C4-
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Fig. 3. A schematic representation of evolution of the maize Pdk1 and Pdk2 genes that encode PPDK. Pdk1 genes of the existing C3 and C4
plants have a dual promoter system and encode the chloroplastic and cytosolic isoforms, while Pdk2 genes have a single promoter and encode the
cytosolic isoform. Chloroplastic and cytosolic genes represent genes that encode the chloroplastic and cytosolic isoforms, respectively.
Evolution and engineering of C4 enzymes 183
Evolution of other C4-speci®c genes
The same evolutionary scenario can be applied to the C4speci®c PEPC gene. The promoter of the maize C4-speci®c
gene (±1212 to +78) directed high-level, mesophyll-cell-
Fig. 4. The promoter of the C4-speci®c gene for the enzyme speci®c
to the bundle sheath cells directs the expression of a reporter gene in
the bundle sheath cells and vascular bundles in rice plants. The 5¢
region (±1447 to +227, an upstream region from the initiation codon)
of the C4-speci®c PEP-CK gene from Zoysia japonica (PEP-CK type)
was fused to the 5¢ side of the GUS gene and introduced into rice
plants by Agrobacterium-mediated gene transfer. Histochemical
localization of GUS activity is shown. Cross-sections of leaf blade (a),
leaf sheath (b) and stem (c). Scale bars 0.1 mm. M Nomura, M
Matsuoka, unpublished results.
speci®c and light-inducible expression of the GUS gene in
transgenic rice (Matsuoka et al., 1994). The C4-speci®c
PPDK and PEPC are both located in the mesophyll cells of
C4 plants. Quite recently, it has been found that C4-speci®c
genes for the enzymes located in the bundle sheath cells
might evolve in similar ways. The promoter of the C4speci®c PEP-CK gene of a turf grass Zoysia japonica
(PEP-CK type) directed speci®c expression of the GUS
gene in the bundle sheath cells and vascular bundles in
transgenic rice (M Nomura, M Matsuoka, unpublished
results; Fig. 4). Similar results have been obtained with the
C4-speci®c gene for the mitochondrial AspAT of Panicum
miliaceum (NAD-ME type; M Nomura, M Matsuoka,
unpublished results). It seems quite likely that, irrespective
of the enzyme location and the subtype of the C4 pathway,
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speci®c PPDK gene might differ from this in the C4 species
of Flaveria. Until now, only Pdk1 with a dual promoter
system, but not Pdk2, has been identi®ed in both C3 and C4
species of Flaveria (Rosche et al., 1994; Rosche and
Westhoff, 1995). If Pdk2 were to be missing, the evolution
in this species probably proceeded without gene duplication.
Modi®cations required for the evolution of the maize
C4-speci®c PPDK gene have been investigated by comparing expression patterns of C4-like and C4-speci®c genes
in rice and maize plants. When a reporter gene (GUS gene)
was expressed in rice plants under the control of the
promoter of the C4-speci®c gene in the maize Pdk1 (±1032
to +71, relative to the transcription initiation site), GUS
was expressed highly speci®cally in the mesophyll cells of
green leaves at a high level and in a light-responsive
manner (Matsuoka et al., 1993). Its expression level was
even higher than that under the control of the cauli¯ower
mosaic virus 35S promoter. On the other hand, when the
GUS gene was expressed in maize plants under the control
of the promoter of the C4-like gene in the rice Pdk1 (±1419
to +512, a region upstream from the initiation codon), GUS
was expressed in both the mesophyll and bundle sheath
cells at low levels, although its expression was light
responsive (Nomura et al., 2000a). These results clearly
show that a cis-acting element(s) for light-responsive
expression is present in the rice promoter, but that those for
cell-speci®c and high-level expression are missing and had
to be acquired during the course of evolution from a C4like to a C4-speci®c gene (Fig. 3). Some of these cis-acting
elements in the promoter of the maize C4-speci®c gene
have been identi®ed (Sheen, 1991; Matsuoka and
Numazawa, 1991; Imaizumi et al., 1997; Nomura et al.,
2000a). Another important implication of the results is that
trans-acting elements (e.g. transcription regulators) required for the expression of the C4-speci®c gene are
present in the leaves of the C3 plant, rice.
The expression pattern of the promoter of the C3speci®c gene in Pdk1, on the other hand, does not differ
much between the maize and rice genes. The C3-speci®c
promoters from the maize and rice Pdk1 both directed
expression of the GUS gene in non-photosynthetic organs
such as grains and roots in transgenic rice (Nomura et al.,
2000b).
Thus, modi®cations of Pdk1 required for the evolution
from a C4-like to a C4-speci®c gene are relatively simple;
namely, gain of the cis-acting elements for cell-speci®c
and high-level expression in the promoter region. As
described later, however, cis-acting elements for highlevel expression are not restricted to the promoter region.
184 Miyao
Table 1. Increase in activities of C4 enzymes in transgenic rice leaves
C4 enzyme
(location in C4 plants)
PEPC (MC)
PPDK (MC)
NADP-ME (BSC)
PEP-CK (BSC)
Introduced construct
Intact maize gene
Rice Cab prom::maize FL C4 cDNA
Maize Pdk1 C4 prom::maize FL C4 cDNA
Intact maize gene
Rice Cab prom::rice FL C3 cDNA
Rice Cab prom::maize FL C4 cDNA
Rice Cab prom::Zoysia FL C4 cDNA
Maize C4 PEPC prom::Urochloa C4 cDNAc
Maize Pdk1 C4 prom::Urochloa C4 cDNAc
Highest enzyme activitya
(increase in fold)
Over rice activity
Over maize activity
110
5
5
40
<5
30
70
±
3±4
<0.1
<0.1
0.5
<0.1
0.6
±
0.1b
±
±
0.5d
0.5d
References
Ku et al., 1999
Fukayama et al., 2001
Fukayama et al., 2001
Fukayama et al., 2001
Tsuchida et al., 2001
Tsuchida et al., 2001
Takeuchi et al., 2000
Miyao M. et al.,
unpublished results
Suzuki et al., 2000
Suzuki et al., 2000
Highest enzyme activities among the primary transgenic plants are listed.
Highest level of the enzyme protein relative to the level in Zoysia leaves is presented.
c
The Urochloa C4-speci®c PEP-CK cDNA was fused to a sequence of the transit peptide for targeting to chloroplasts.
d
Highest activities of the secondary transgenic plants relative to the activity of Urochloa leaves are presented.
MC, mesophyll cells; BSC, bundle sheath cells; prom, promoter; FL, full-length.
b
gene modi®cations for the evolution of C4-speci®c genes
followed similar mechanisms.
How to overproduce C4 enzymes in the
mesophyll cells of C3 plants
In leaves of C3 plants, photosynthesis and subsequent
carbon and nitrogen metabolism proceed mainly in the
mesophyll cells. To alter carbon metabolism in leaves of
C3 plants, C4 enzymes have to be overproduced in these
cells. From the evolutionary origins of the C4-speci®c
genes and the expression patterns of their promoters in the
leaves of C3 plants, it was anticipated that the introduction
of the intact C4-speci®c genes into C3 plants would lead to
high-level and cell-speci®c expression of C4 enzymes. In
fact, this strategy was effective in overproducing C4
enzymes speci®c to the mesophyll cells of C4 plants. By
contrast, to overproduce enzymes speci®c to the bundle
sheath cells, conventional techniques with gene constructs
containing a strong promoter fused to a cDNA were
effective (Table 1).
Enzymes located in the mesophyll cells of C4 plants
The ®rst trial of this kind was conducted with the intact
maize PEPC gene expressed in transgenic rice plants (Ku
et al., 1999). The maize gene of 8.8 kb that contained all
exons and introns and its own promoter and terminator
sequences was introduced into rice plants. As expected, the
activity of PEPC in the leaf protein extract was greatly
increased up to 110-fold that of non-transformants or 3fold the maize activity. The level of the PEPC protein
accounted for 12% of total leaf soluble protein at most. The
introduction of the intact maize gene was also effective in
overproducing PPDK in rice leaves (Fukayama et al.,
2001). The introduction of the intact maize Pdk1 of 7.3 kb
increased the PPDK activity in rice leaves up to 40-fold
that of non-transformants or about half of the maize
activity. In a homozygous transgenic line, the PPDK
protein accounted for 35% of total leaf soluble protein or
16% of total leaf nitrogen, much above the levels of
foreign protein in transgenic plants reported previously.
The C4-speci®c gene of the maize Pdk1 was exclusively
expressed in the leaves of these transgenic rice plants while
the C3-speci®c gene was expressed in grains, an indication
that the maize Pdk1 is expressed in rice plants with an
organ speci®city similar to that in maize plants.
To examine whether or not the promoter sequence of the
C4-speci®c gene is suf®cient for the high-level expression,
the full-length cDNA encoding the maize C4-speci®c
PPDK was expressed under the control of the promoter of
the C4-speci®c gene of the maize Pdk1 or the rice Cab
promoter (Fukayama et al., 2001). Cab encodes the lightharvesting chlorophyll-binding protein and is expressed at
high levels in photosynthetically active organs (Sakamoto
et al., 1991). The introduction of these gene constructs,
however, increased the activity of PPDK in rice leaves
only up to several fold that of non-transformants
(Fukayama et al., 2001). Thus, the transcriptional activity
of the C4-speci®c promoter cannot be the prime reason for
high-level expression. The 5¢- and 3¢-noncoding regions by
themselves cannot lead to high-level expression either,
since the constructs containing the full-length cDNA with
these regions were not effective. Therefore, it is quite
possible that, in addition to the promoter region, the
presence of introns or the terminator sequence, or a
combination of both, is required for high-level expression.
In transgenic rice plants containing the C4-speci®c
PEPC or PPDK gene, the levels of transcripts and protein
and the activity of C4 enzyme in the leaves all correlated
well with the copy number of the introduced gene (Ku et al.,
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a
Evolution and engineering of C4 enzymes 185
Enzymes located in the bundle sheath cells of C4
plants
Since the intact C4-speci®c genes for these enzymes would
be expressed speci®cally in the bundle sheath cells of C3
plants, they cannot be used for overproduction in
photosynthetically active mesophyll cells. More conventional techniques were applied and have proven successful.
The expression of the maize C4-speci®c NADP-ME cDNA
under the control of the rice Cab promoter increased the
activity of NADP-ME in rice leaves to 30- or 70-fold that
of non-transformants (Takeuchi et al., 2000; Tsuchida
et al., 2001). The level of the NADP-ME protein was also
increased up to several per cent of total leaf soluble
protein. Such high-level expression was unique to the
cDNA for the C4-speci®c NADP-ME, and the expression
of the cDNA for the rice C3-speci®c isoform under the
control of the same promoter increased the activity only
some fold (Tsuchida et al., 2001). This observation
suggests that expression of the rice C3-speci®c NADPME is suppressed at co- and/or post-transcriptional levels
by some regulation mechanisms intrinsic to rice, while that
of the foreign C4-speci®c isoform can escape from such
suppression. The Zoysia C4-speci®c PEP-CK was also
overproduced by introduction of a cDNA construct (M
Miyao et al., unpublished results).
Overproduction of C4 enzymes in a different
intracellular compartment
The C4 enzymes described above were all overproduced in
the same intracellular compartment in C3 plants as in C4
plants, namely, PEPC and PEP-CK in the cytosol and
PPDK and NADP-ME in the chloroplasts. The intracellular location of foreign enzyme can be altered by use of
targeting signals. To overproduce PEP-CK in the chloroplasts of the mesophyll cells of rice leaves, the cDNA of
the C4-speci®c PEP-CK from Urochloa panicoides was
fused to a sequence of the transit peptide for targeting to
chloroplasts, and expressed under the control of the maize
C4-speci®c PEPC or PPDK promoter (Suzuki et al., 2000).
The PEP-CK activity of transgenic rice leaves reached
about half of that in the Urochloa leaves. Similarly,
bacterial enzymes were overproduced in the chloroplasts
of transgenic C3 plants (HaÈusler et al., 2001; Panstruga
et al., 1997).
Factors affecting the expression levels of transgenes
In general, expression of transgenes is hampered by many
mechanisms including the positional effects (Gelvin,
1998), silencing (Gallie, 1998; Chandler and Vaucheret,
2001) and rearrangement (Hiei et al., 1994) of transgenes.
During the course of the study of overproducing C4
enzymes, it was found that the rearrangement occurs
frequently during the gene transfer mediated by
Agrobacterium tumefaciens. A signi®cant fraction of
transgenic rice plants introduced with the intact maize
C4-speci®c gene showed activities of C4 enzymes comparable to or even lower that that of non-transformants (Ku
et al., 1999; Fukayama et al., 2001). DNA gel-blot analysis
of these low-expressing lines showed that transgenes in all
lines tested sustained partial deletion and/or chimeric
linking (Fukayama et al., 2001). Such rearrangement is not
peculiar to long transgenes with complex exon-intron
structures, and it did occur in ®ve out of nine transgenic
rice plants introduced with a cDNA construct of 4.4 kb
(Miyao et al., 2001). It is possible that cis-acting elements
and/or the transit sequence are selectively deleted from an
introduced gene, altering the level and/or location of a C4
enzyme in transgenic C3 plants.
As described above, overproduction of C4 enzymes in
C3 plants can be achieved by introducing appropriate gene
constructs. It is also necessary to screen a number of
transgenic plants to obtain a desired expression level of a
C4 enzyme and to con®rm the enzyme location in the
leaves of C3 plants.
Regulation and physiological impacts of C4
enzymes overproduced in C3 plants
PEPC
The activity of PEPC in higher plants is regulated by two
different mechanisms; one the reversible protein phos-
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1999; Fukayama et al., 2001). It was also found that the
levels of transcripts in the leaves per copy of the maize C4speci®c gene were comparable in both maize and transgenic rice plants (Ku et al., 1999; Fukayama et al., 2001).
As described above, the promoters of C4-speci®c PPDK
and PEPC genes have the cis-elements for organ- and cellspeci®c expression. It is likely that the maize C4-speci®c
genes behave in a qualitatively and also quantitatively
similar way in both maize and transgenic rice plants.
Overproduction by the introduction of the intact C4speci®c gene, however, seems to have some limitation in
that transgenes from phylogenetically closely related
plants have to be used to achieve high-level expression
of the C4 enzyme in C3 plants. The intact maize C4-speci®c
PEPC gene was not expressed at high levels in tobacco
leaves, because of incorrect transcription initiation
(Hudspeth et al., 1992). Not only incorrect initiation and
termination of transcription, but also incorrect splicing
could occur when genes from monocots are introduced into
dicots (Goodall and Filipowicz, 1991). Thus, phylogenetic
distance may hamper the expression of genes from C4
plants in the leaves of C3 plants.
Conventional techniques for overproduction of transgenes,
namely, the introduction of a chimeric gene containing
cDNA for a C4 enzyme, fused to a strong promoter alone or
together with enhancer sequences, can also increase the
activity of C4 enzymes in the leaves of C3 plants, although
the increase does not exceed several fold that of nontransformants (for a review see Matsuoka et al., 2001).
186 Miyao
an anaplerotic function of the C3-speci®c PEPC, which
replenishes the tricarboxylic acid cycle with organic acids
to meet the demand of carbon skeletons for amino acid
synthesis (Champigny and Foyer, 1992). The effects on
stomatal movement were observed only under non-steadystate conditions. Transient closure of stomata at the onset
of gas-exchange measurements and after a step increase in
light intensity were reported in the transgenic potato and
rice, respectively. Accelerated stomatal opening, by contrast, was observed in the transgenic potato, but not in the
transgenic rice. PEPC has long been implicated in the
synthesis of malate as an osmotically active solute in
stomata (Assmann, 1993). At present, it remains obscure if
foreign PEPC is expressed in guard cells of these
transformants.
In view of the critical function of PEPC in photosynthesis of C4 and CAM plants, some researchers had
expected that overproduction of PEPC alone would
improve the photosynthetic performance of C3 plants. A
research group claims that the photosynthetic rate under
saturating light can be greatly increased by overproduction
of the maize PEPC in transgenic rice plants (Jiao et al.,
2001). Their results, however, require reconsideration,
since the correlation between the photosynthetic rate and
the level of the PEPC protein in transgenic rice leaves has
not yet been con®rmed. Other groups all reported negative
effects on photosynthesis, and the photosynthetic rate was
slightly lowered by the overproduction. It has previously
been reported that the O2 inhibition of photosynthesis was
mitigated by overproduction of the maize PEPC, and
suggested that the maize PEPC may participate in
photosynthetic CO2 ®xation in transgenic rice leaves (Ku
et al., 1999). Later experiments, however, indicated that
the initial CO2 ®xation product, determined by 14CO2
labelling experiments with transgenic rice plants showing
a 50-fold elevation in extractable PEPC activity, was
exclusively the C3 compound 3-phosphoglycerate
(Fukayama et al., 2002). The apparent reduction of O2
inhibition can be explained by more marked suppression of
photosynthesis by overproduction of PEPC at 2% O2 than
at 21% O2 (Matsuoka et al., 2000). In a recent paper
(Agarie et al., 2002), it has been proposed that the lower
photosynthetic rate resulted from the reduced capacity of
regeneration of inorganic phosphate. However, a signi®cant reduction of the photosynthetic rate was observed at
very low intercellular CO2 concentrations and a low O2
concentration where the Rubisco activity but not the
phosphate regeneration limits photosynthesis (Fukayama
et al., 2002). It is more likely that the suppression of
photosynthesis results from the enhanced respiration by
elevated PEPC.
Transgenic Arabidopsis overproducing PEPC from a
cyanobacterium Synechococcus vulcanus has recently
been reported (Chen and Izui, 2002). The Synechococcus
PEPC has some advantages for raising the in vivo activity
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
phorylation of a conserved serine residue near the Nterminus, and the other through various metabolite
effectors such as glucose-6-phosphate (Glc6P), malate,
aspartate, and glutamate (Vidal and Chollet, 1997). Upon
phosphorylation, PEPC becomes more sensitive to the
activator Glc6P and less sensitive to the feedback inhibitor
malate, being more active in vivo (Vidal and Chollet,
1997). The phosphorylation itself is also inhibited by
malate through the conformational change of PEPC
(Bakrim et al., 1998). In leaves of C4 plants, PEPC is
phosphorylated in the light and dephosphorylated in
darkness and its activity is modulated in response to
changes in light intensity (Vidal and Chollet, 1997). The
maize PEPC expressed in transgenic rice leaves also
underwent activity regulation via phosphorylation, but in
an opposite manner (Fukayama et al., 2002). It remained
dephosphorylated and less active during the daytime and
became phosphorylated and more active in the night in
transgenic rice leaves. Since the activity of the endogenous
rice PEPC was also down-regulated during the daytime, it
is likely that both the maize and rice PEPC undergo
activity regulation by the same mechanisms in rice leaves.
Bacterial PEPC lacks the phosphorylation site (Vidal and
Chollet, 1997) and can escape from down-regulation via
phosphorylation.
Another issue affecting potential activity of foreign
PEPC in the leaves C3 plants is the cytosolic concentrations of potential inhibitors and activators. The concentrations of inhibitors of higher plant PEPC are high in the
cytosol of the mesophyll cells of C3 plants, about 1 mM for
malate and around 40 mM for aspartate and glutamate
(Heineke et al., 1991). Bacterial PEPC is also inhibited by
aspartate, and in addition, it requires acetyl-CoA as an
activator (Chen et al., 2002). Taken together, the actual
in vivo activity of foreign PEPC, either from higher plants
or bacteria, in the leaves of transgenic C3 plants is lower
than maximum extractable activities, especially when
measured in the presence of activators.
Until now, several transgenic C3 plants overproducing
PEPC have been produced and analysed precisely
(Matsuoka et al., 2001). All the transformants analysed
so far show a higher level of malate (Hudspeth, et al.,
1992; Kogami et al., 1994; HaÈusler et al., 1999) or OAA
(Fukayama et al., 2002) in the leaves compared to that of
non-transformants, an indication that foreign PEPC is
partly active in these transformants. Physiological impacts
of the elevated PEPC activity reported to date have varied
with no clear consensus. Among these, stimulation of
respiration in the light and destabilization of stomatal
opening have been observed in different plant species
overproducing PEPC of different origin, namely, transgenic potato overproducing PEPC from Corynebacterium
glutamicum (Gehlen et al., 1996) and transgenic rice
overproducing the maize C4-speci®c PEPC (Fukayama
et al., 2002). The stimulated respiration is consistent with
Evolution and engineering of C4 enzymes 187
PPDK
In leaves of C4 plants, the activity of PPDK is rapidly
modulated in response to changes in light intensity by
reversible protein phosphorylation, which is mediated by a
bifunctional regulatory protein (Burnell and Hatch, 1985).
Unlike phosphorylation of PEPC, PPDK is dephosphorylated in the light and dephosphorylated in darkness, and
upon phosphorylation it is completely inactivated. Such a
strict activity regulation is a prerequisite for proper
operation of the C4 pathway since the synthesis of PEP
by PPDK consumes two molecules of ATP. The activity of
the maize C4-speci®c PPDK expressed in rice leaves was
also light±dark regulated as it is in maize, being upregulated in the light (Fukayama et al., 2001).
There are four reports on transgenic plants, which
overproduce PPDK derived from higher plants; transgenic
Arabidopsis (Ishimaru et al., 1997), potato (Ishimaru et al.,
1998), rice (Fukayama et al., 2001) overproducing the
maize C4-speci®c PPDK, and transgenic tobacco overproducing PPDK from a CAM plant Mesembryanthemum
crystallinum (Sheriff et al., 1998). Physiological impacts
were minimal and no changes in the photosynthetic
characteristics were observed in these transformants,
even in the transgenic rice with a 40-fold increase in
activity (Fukayama et al., 2001). In general, the reaction of
PPDK is freely reversible, depending on concentrations of
substrates, activators, and inactivators (Burnell and Hatch,
1985). This could be the reason why the overexpression of
PPDK does not result in signi®cant effects on carbon
metabolism in the leaves.
NADP-ME
In contrast to PEPC and PPDK, there is no speci®c
regulatory protein for higher plant chloroplastic NADPME. The activity of the C4-speci®c NADP-ME is increased
under illumination through increases in pH and concentration of Mg2+ in the chloroplast stroma (Edwards and
Andreo, 1992).
Two sets of transgenic rice plants overproducing the
maize C4-speci®c isoform (Takeuchi et al., 2000; Tsuchida
et al., 2001) and the rice C3-speci®c isoform of NADP-ME
(Tsuchida et al., 2001) have been reported. The transformants overproducing the rice enzyme with some fold
increase in activity did not show any detectable differences
in their growth, while those overproducing the maize
enzyme at the same activity level showed serious stunting
and bleaching of leaf colour, due to enhanced photoinhibition of photosynthesis under natural light conditions. It is
proposed that the action of the maize NADP-ME in the
chloroplasts increases the NADPH/NADP ratio and suppresses photorespiration, rendering photosynthesis more
susceptible to photoinhibition (Takeuchi et al., 2000;
Tsuchida et al., 2001). The C4-speci®c NADP-ME has a
higher Vm value, lower Km values for substrates, and
higher optimum pH, as compared with the C3-speci®c
isoform (Casati et al., 1997). Such features are suitable for
strict regulation of the enzyme activity in the bundle sheath
cell chloroplasts of C4 plants, but they allow the enzyme to
continue operating in the leaves of C3 plants even when
serious damage occurs.
Future applications of overproduction of C4
enzymes
A major objective of overproduction of C4 enzymes in C3
plants is to improve the photosynthetic performance. As
described above and reviewed recently (HaÈusler et al.,
2002), none of the positive effects on photosynthesis have
been observed in transgenic C3 plants overproducing a
single C4 enzyme. Transgenic C3 plants overproducing
multiple enzymes are being produced and analysed in
some research groups (HaÈusler et al., 2002). Although the
introduction of the `C4-like' pathway into the mesophyll
cells of C3 plants is one strategy being adopted (Mann,
1999; Surridge, 2002), whether or not this pathway can
operate with desirable effects on C3 photosynthesis is a
matter of controversy (Edwards, 1999; Leegood, 2002;
HaÈusler et al. 2002). Considering the C4 pathway operating
in a single cell found in some aquatic organisms (for a
review see Leegood, 2002), it might be possible that the
C4-like pathway could support C3 photosynthesis under
some stress conditions such as drought, in which the CO2
availability is limited.
Apart from photosynthesis, overproduction of a single
C4 enzyme seems to have some positive effects on
physiology of C3 plants. It has been reported that
overproduction of the chloroplastic, but not cytosolic,
PPDK increased the number of seeds per seed capsule and
the weight of each seed capsule in transgenic tobacco
(Sheriff et al., 1998), and that overproduction of the maize
C4-speci®c PEPC improved resistance to aluminium of
root elongation in transgenic rice (Miyao et al., 2001). Of
course, it is of prime importance to elucidate mechanisms
for these effects and to con®rm whether or not similar
phenomena can be generally observed in different plant
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
of PEPC in the leaves of C3 plants. It does not undergo
activity regulation via phosphorylation or, in contrast to
other bacterial PEPCs, is not inactivated by malate, and
moreover, it does not require acetyl-CoA for activation
(Chen et al., 2002). Expression of this PEPC in
Arabidopsis led to stunting and bleaching of leaf colour
(Chen and Izui, 2002). Similar phenomena were observed
in transgenic potato overproducing the potato enzyme that
had been modi®ed for a higher af®nity for PEP and
lowered sensitivity toward malate (Rademacher et al.,
2002). It has been demonstrated that carbon ¯ow in these
transgenic potato plants was redirected from soluble sugars
and starch to organic acids and amino acids (Rademacher
et al., 2002).
188 Miyao
species. Taking account of a variety of housekeeping
functions of the C3-speci®c enzymes, it is not unlikely that
overproduction of C4 enzymes could improve various
features of C3 plants.
Acknowledgements
The author is grateful to Professor Makoto Matsuoka, Nagoya
University, Japan, and Professor RE HaÈusler, University of KoÈln,
Germany, for providing unpublished information, and to Ms Hiroko
Tsuchida for her assistance in preparing the manuscript.
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