Journal of Experimental Botany, Vol. 62, No. 9, pp. 3021–3029, 2011
doi:10.1093/jxb/err023 Advance Access publication 31 March, 2011
REVIEW PAPER
Lessons from engineering a single-cell C4 photosynthetic
pathway into rice
Mitsue Miyao1,*, Chisato Masumoto1, Shin-Ichi Miyazawa1 and Hiroshi Fukayama2
1
Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences (NIAS), Kannondai, Tsukuba 305-8602,
Japan
2
Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
* To whom correspondence should be addressed. E-mail: [email protected]
Received 15 November 2010; Revised 16 January 2011; Accepted 19 January 2011
Abstract
The transfer of C4 plant traits into C3 plants has long been a strategy for improving the photosynthetic performance
of C3 plants. The introduction of a pathway mimicking the C4 photosynthetic pathway into the mesophyll cells of C3
plants was only a realistic approach when transgenic technology was sufficiently well developed and widely
adopted. Here an attempt to introduce a single-cell C4-like pathway in which CO2 capture and release occur in the
mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is
described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and
12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced
and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these
studies nonetheless have important implications for the evolution of C4 photosynthetic genes and their metabolic
regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes
the lessons learned during these attempts to engineer single-cell C4 rice.
Key words: C4 photosynthesis, metabolic engineering, NADP-malate dehydrogenase, NADP-malic enzyme,
phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, transgenic rice.
Introduction
Many important crops, such as rice (Oryza sativa L.), wheat
(Triticum aestivum L.), soybean (Glycine max L.), and
potato (Solanum tuberosum L.), are classified as C3 plants.
This plant group assimilates atmospheric CO2 directly
through the C3 photosynthetic pathway, which is also called
the Calvin cycle or the photosynthetic carbon reduction
cycle. In contrast, C4 plants such as maize (Zea mays L.),
sorghum [Sorghum bicolor (L.) Moench], and sugarcane
(Saccharum officinarum L.) evolved from C3 plants, acquiring the C4 photosynthetic pathway in addition to the C3
pathway. The C4 pathway acts to concentrate CO2 at the
site of the reactions of the C3 pathway, and thus inhibits
photorespiration (Hatch, 1987). This CO2-concentrating
mechanism enables C4 plants to achieve higher photosyn-
thetic capacity and higher water- and nitrogen-use efficiency
than C3 plants. Since the discovery of C4 photosynthesis
and its agronomic advantages, the transfer of C4 traits to C3
plants has been one strategy for improving the photosynthetic performance of C3 plants. This strategy was initially
attempted by means of conventional hybridization between
C3 and C4 plants (reviewed in Brown and Bouton, 1993)
and more recently using transgenic techniques (reviewed in
Matsuoka et al., 2001; Häusler et al., 2002; Miyao, 2003).
The C4 pathway consists of three key steps: (i) initial
fixation of CO2 by phosphoenolpyruvate carboxylase (PEPC)
to form a C4 acid; (ii) decarboxylation of a C4 acid to release
CO2 near the site of the Calvin cycle; and (iii) regeneration of
the primary CO2 acceptor phosphoenolpyruvate (PEP) by
Abbreviations: BSC, bundle sheath cell; GUS, b-glucuronidase; MC, mesophyll cell; 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-1,5-bisphosphate carboxylase/oxygenase.
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
3022 | Miyao et al.
pyruvate, orthophosphate dikinase (PPDK) (Hatch, 1987).
In terrestrial C4 plants, CO2 release from C4 acids and the
resulting elevation of cellular CO2 levels take place at a site
that is physically separated from the site of the initial
carboxylation. This separation occurs through the bundle
sheath cells (BSCs) and mesophyll cells (MCs) in typical C4
plants (Hatch, 1987), and through two distant subcellular
compartments in the recently discovered single-cell C4 plants
(Edwards et al., 2004). Structural and biochemical features of
the chloroplasts at these two sites are also different. It has
been proposed that such compartmentation and chloroplast
differentiation, together with structural adaptation to minimize CO2 diffusion away from the CO2 release site, are
essential for C4 photosynthesis (Hatch, 1987; Edwards et al.,
2004). The molecular basis for such structural and chloroplast differentiation is not yet fully understood, but is a hot
topic in current C4 photosynthesis research.
Another example of C4 photosynthesis has been found in
aquatic plants, in which C4 photosynthesis is accomplished
in a single cell without any compartmentation and chloroplast differentiation (Bowes et al., 2002; Fig. 1A). At least
three submerged macroalgae, all of which are freshwater
monocots, perform C4 photosynthesis in this manner
(Bowes et al., 2002). Hydrilla verticillata (L.f.) Royle has
been the best documented. It is a facultative C4 plant that
shifts from C3 to C4 photosynthesis under low CO2
conditions without undergoing any structural modifications
of its leaf cells. During the shift to C4 photosynthesis, genes
encoding the C4-specific isozymes of PEPC, PPDK, and
NADP-malic enzyme (NADP-ME) are upregulated (Rao
et al., 2002, 2006; Estavillo et al., 2007).
Because of the simplicity of the Hydrilla system, a number
of attempts have been made to introduce this type of C4-like
pathway into the MCs of C3 plants (reviewed in Matsuoka
et al., 2001; Häusler et al., 2002; Miyao, 2003). Other C4like pathways consisting of two enzymes [PEPC and
phosphoenolpyruvate carboxykinase (PEP-CK); Suzuki
et al., 2006; Fig. 1B] or of five enzymes [PEPC, PPDK or
PEP synthetase, PEP-CK, NADP-malate dehydrogenase
(NADP-MDH), and NADP-ME; Häusler et al., 2002] have
also been proposed. The key steps in these pathways are the
carboxylation of PEP by PEPC in the cytosol and decarboxylation of the resultant C4 acid inside the chloroplast.
Whether or not such pathways can operate with desirable
effects for C3 photosynthesis has been a matter of controversy (Edwards, 1999; Häusler et al., 2002; Leegood, 2002).
Two major concerns have been raised. One is whether
a closed cycle is created without any modification of
intracellular metabolite transport. All the proposed pathways involve the import of oxaloacetate (OAA) into and the
export of PEP from the chloroplasts (Fig. 1). The import of
OAA has not been a subject of debate, since OAA is
actively taken up by the chloroplast via the malate valve,
which functions in transferring reducing equivalents from
the chloroplast stroma to the cytosol (Scheibe, 2004) when
chloroplasts perform photosynthesis under illumination. In
addition, the envelope of the C3 MC chloroplast has a high
capacity to translocate dicarboxylic acids (Bräutigam and
Fig. 1. Introduction of the C4-like pathway into the MCs of C3
plants. (A) The C4-like pathway of H. verticillata, consisting of
PEPC, PPDK, NADP-MDH, and NADP-ME. Previously, the activity
of NADP-MDH in the C3 mesophyll chloroplasts was believed to
be sufficiently high that the C4-like pathway would operate if
PEPC, PPDK, and NADP-ME could be overproduced. (B) The C4like pathway consisting of PEPC and PEP-CK (Suzuki et al., 2006).
The Hydrilla pathway (A) consumes two molecules of ATP (one
consumed by PPDK and the other required for the conversion of
AMP produced by PPDK into ADP), whereas the PEPC+PEP-CK
pathway (B) consumes one molecule of ATP. CA, carbonic
anhydrase; Mal, malate; Pyr, pyruvate.
Weber, 2011). In contrast, it has long been argued that the
export of PEP should limit this pathway, since the transport
activity of the chloroplast envelope for PEP is low in the
leaves of C3 plants (e.g. see Fischer et al., 1997). The second
concern is whether CO2 released inside the chloroplast can
be retained long enough to promote photosynthesis or
would be lost to the atmosphere (Leegood, 2002). Mathematical modelling of the Hydrilla-type C4-like pathway
suggested that for elevation of the CO2 concentration to
occur inside the chloroplast, the activity of the C4-like
pathway must reach at least 38% of the maximal carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) under atmospheric CO2 conditions (von
Caemmerer, 2003).
Here an attempt to introduce the Hydrilla-type C4-like
pathway into the MCs of rice by overproducing C4
photosynthetic enzymes from C4 grasses is described. Each
of the four C4 enzymes (i.e. PEPC, PPDK, NADP-MDH,
and NADP-ME) have been successfully overproduced in
rice leaves (Ku et al., 1999; Fukayama et al., 2001; Tsuchida
et al., 2001; Taniguchi et al., 2008). Among the 12 different
sets of transgenic rice that overproduce the four enzymes,
independently or in combination, only quadruple transformants that overproduce all four showed increased
photosynthesis rates (Taniguchi et al., 2008). At present,
however, this enhanced photosynthesis does not appear to
Engineering a single-cell C4 pathway into rice | 3023
result from a CO2-concentrating function of the introduced
pathway. Although these results are disappointing, nonetheless during the course of this research unexpected findings
were made that have important implications for the
evolution of C4 photosynthetic genes, regulation of the
activity of C4 enzymes in C3 plants, and metabolic
engineering of rice. In this article, these findings are
presented.
Lesson 1: sites of overproduction of C4
enzymes in rice leaves
To overproduce PEPC and PPDK, MC-specific enzymes of
C4 plants, intact maize C4-specific genes for these enzymes,
each containing its own promoter and terminator sequences
and exon–intron structure were introduced. This strategy
was based on observations that the 5’-flanking region of the
maize C4-specific genes (–1212 to +78 bp and –1032 to +71
bp from the transcription start site for the maize PEPC and
PPDK genes, respectively) drove high-level and MC-specific
expression of a reporter b-glucuronidase (GUS) gene in rice
leaves (Matsuoka et al., 1993, 1994). As expected, the intact
maize genes were strongly expressed in the transgenic rice
leaves, and the maize PEPC and PPDK proteins accounted
for up to 12% and 35%, respectively, of total leaf soluble
protein (Ku et al., 1999; Fukayama et al., 2001). Comparison of the PPDK protein levels among transgenic rice
plants with three different introduced gene constructs
(Fukayama et al., 2001) indicated that, in addition to the
5’-flanking region, one or more introns or the terminator
sequence of the maize genes, or a combination of both, was
necessary for high-level expression of the maize genes.
Contrary to expectations, however, immunoelectron microscopy subsequently revealed that the maize PPDK protein accumulated in both MCs and BSCs of rice leaves
(Fig. 2). Based on the densities of gold particles, the levels
of the maize PPDK protein in the chloroplasts do not differ
greatly between the two cell types. It is likely that faint
GUS staining in the BSCs was underestimated because of
the much denser staining in the surrounding MCs in
a previous promoter::GUS analysis using the maize PPDK
gene (Matsuoka et al., 1993). However, even if the
expression is not specific to the MCs, the majority of
the maize PPDK protein could be located in these cells if
the distribution of the chloroplasts between the MCs and
BSCs is taken into account; >90% of the total number of
chloroplasts are found in the MCs of rice leaves (Yoshimura
et al., 2004). At present, the possibility that maize PEPC
might also accumulate in both cell types of transgenic rice
leaves cannot be ruled out.
Information on the expression of BSC-specific C4 enzyme
genes in the leaves of C3 plants was unavailable when these
studies began. A conventional technique for overproducing
foreign proteins was therefore employed, in which a fulllength cDNA was expressed under the control of the rice
Cab promoter, which directed strong and light-regulated
expression of a reporter gene in leaf chlorenchyma cells
(Sakamoto et al., 1991). The expression of the cDNA for
the maize C4-specific NADP-ME in this way resulted in up
to 2000% elevation of NADP-ME activity in rice leaves
(Tsuchida et al., 2001). The rice C3-specific NADP-ME and
the sorghum NADP-MDH were overproduced in rice leaves
in the same way. These foreign enzymes might be present in
both MCs and BSCs of rice leaves, both of which have
chloroplasts (see Fig. 2).
The evolutionary scenario inferred from the previous
promoter::GUS analyses of the maize PEPC and PPDK
genes was relatively simple: in this scenario, maize acquired
one or more cis elements for high-level MC-specific
expression of the genes, whereas mechanisms for such
expression, including one or more trans factors, were
already present in rice leaves (Matsuoka, 1995; Nomura
et al., 2000). This scenario was considered for BSC-specific
C4 enzyme genes, since the promoter::GUS analyses of the
two different genes revealed GUS expression in the BSCs:
the 5’-flanking region of the C4-specific NADP-ME gene of
C4 Flaveria bidentis (L.) Kuntze conferred preferential
expression of GUS in the BSCs of transgenic F. bidentis
(Marshall et al., 1997), and that of the PEP-CK gene of
Zoysia japonica Steud. led to strong and specific expression
of GUS in the BSCs and vascular bundles of transgenic rice
(Miyao, 2003). However, subsequent analyses using rice
plants (Nomura et al., 2005a, 2005b) have demonstrated
that the 5’-flanking regions of BSC-specific C4 genes follow
Fig. 2. Accumulation of the maize C4-specific PPDK protein in the
chloroplasts of (A) the MC and (B) the BSC of transgenic rice
leaves. Immunogold labelling was used to locate PPDK in transgenic rice carrying the intact maize C4-specific PPDK gene (line
PD259; Fukayama et al., 2001; bars ¼ 1 lm). Ultrathin sections
were taken from the leaves and labelled with antiserum raised
against maize C4-specific PPDK following the method of Ueno
(2004). This antiserum predominantly cross-reacted with maize
PPDK in the leaves of PD259 (Fukayama et al., 2001). The density
of gold particles in the chloroplast was comparable between the
MC and the BSC. Electron micrographs were provided by
Professor Osamu Ueno (Kyushu University, Japan).
3024 | Miyao et al.
the expression patterns of their respective rice orthologues
and do not always confer BSC-specific expression of a reporter
gene in the rice leaves. Unlike the Zoysia PEP-CK gene,
GUS staining was detected in almost all tissues for the
mitochondrial aspartate aminotransferase gene from Panicum
miliaceum L. and the maize NADP-ME gene in transgenic rice
leaves (Nomura et al., 2005a, 2005b). These observations
suggest that the one or more presumed cis elements are not
sufficient for BSC-specific expression of C4 genes in the leaves
of C3 plants.
Recent studies have made much progress in identifying
cis elements in C4 photosynthetic genes (reviewed in
Hibberd and Covshoff, 2010). The promoter-deletion studies of the GLDPA gene for the P-subunit of glycine
decarboxylase, a BSC-specific gene of C4 Flaveria trinervia
(Spreng.) C. Mohr (Engelmann et al., 2008), identified two
regions in its 5’-flanking region, one (–1571 to –1138 bp
from the translation start site) functioning as a transcriptional enhancer and the other (–1138 to –926 bp) involved
in ‘repression’ of gene expression in MCs. A region
proximal to the translation start site (–298 to –1 bp) could
be involved in BSC-specific expression and, in combination
with the region for repression in MCs, it conferred BSCspecific expression of a reporter gene in C4 F. bidentis.
Similar results have been reported for two MC-specific
enzyme genes of C4 Cleome gynandra L.: the genes for
PPDK and for plasma membrane-bound carbonic anhydrase (K. Kajala and J. M. Hibberd, University of Cambridge, unpublished data). One or more general
transcriptional enhancer elements are present in the 5’flanking region, whereas the 5’ and 3’ untranslated regions,
either independently or in combination, are sufficient for
the MC-specific expression of a reporter gene in C4 Cleome
but not in Arabidopsis (Arabidopsis thaliana L.). The latter
observation suggests that post-transcriptional regulation is
involved in the cell specificity of the C4 enzymes in C4
plants. All of this evidence taken together suggests that
mechanisms for cell-specific expression of C4 photosynthetic
genes are more complex than previously expected, and that
not only cis elements but also mechanisms for strict cell
specificity of gene expression must have developed during
the evolution of C4 plants.
Lesson 2: kinetic properties of the
introduced enzymes are important
Overproduction of maize C4-specific NADP-ME, a chloroplastic enzyme, in rice leaves led to enhanced photoinhibition of photosynthesis, bleaching of leaf colour, and serious
stunting, even when the activity of the maize enzyme was
two or three times higher than that in non-transgenic rice
(Tsuchida et al., 2001). These detrimental effects were
ascribed to an increase in the NADPH to NADP+ ratio in
the chloroplast stroma and to suppression of photorespiration due to depletion of cellular malate, which must be
exported from the chloroplast in exchange for 2-oxoglutarate
during photorespiration. A significant reduction in the leaf
malate level was observed in transgenic Arabidopsis in which
cDNA for the maize C4-specific NADP-ME was expressed
under the control of the cauliflower mosaic virus 35S
promoter (Fahnenstich et al., 2007). In contrast to the maize
NADP-ME, overproduction of the rice C3-specific isozyme,
at activities up to five times that in non-transgenic rice, did
not affect either photosynthesis or growth of rice plants
(Taniguchi et al., 2008).
Kinetic studies using recombinant maize proteins
showed that a major difference between C3-specific (nonphotosynthetic) and C4-specific NADP-MEs was found in
the Km value for NADP+, which was 70 and 8 lM for the
C3- and C4-specific isozymes, respectively (Saigo et al.,
2004). The NADPH to (NADP++NADPH) ratio in the
chloroplast stroma ranges from 0.35 to 0.70, and is higher
under illumination (Fridlyand et al., 1998). It is likely that
the C4-specific isozyme’s high affinity for NADP+ allows it
to sustain the reaction in the direction of decarboxylation of
malate, even when the NADP+ concentration in the stroma
is low under illumination. Transport of dicarboxylic acids
across the chloroplast envelope of C3 leaves is mediated by
antiporters that transport molecules along their concentration gradients (Bräutigam and Weber, 2011). Therefore, the
consumption of the stromal malate by the maize enzyme
leads to uptake of malate from the cytosol, though malate
must be exported from the stroma as a carrier of reducing
power under illumination (Scheibe, 2004).
Because of the detrimental effects of maize C4-specific
NADP-ME on rice plants, the rice C3-specific isozyme was
used for the introduction of the C4-like pathway into rice,
although the elevation of leaf NADP-ME activity was <5fold that of non-transgenic rice (Tsuchida et al., 2001).
With the rice enzyme, care should be taken to shift its
reaction equilibrium towards decarboxylation of malate,
since a high NADPH to (NADP++NADPH) ratio in the
stroma under illumination promotes carboxylation of pyruvate by the C3-specific isozyme, whose affinity for NADP+ is
low.
Studies of the NADP-ME gene family in Arabidopsis
(Gerrard Wheeler et al., 2005; Brown et al., 2010) have
demonstrated that, among the four isozymes, the cytosolic
NADP-ME2 protein that is mainly located in leaf veins
contributes >80% of total NADP-ME activity in the rosette
leaves. The chloroplastic NADP-ME4, an orthologue of the
C4-specific enzyme, thus accounts for <20% of the total leaf
activity of NADP-ME. In addition, the NADP-ME4 gene is
expressed in both MCs and veins. When cDNA for the
chloroplastic isozyme is expressed under the control of the
Cab promoter, the resulting 200% increase in leaf NADPME activity very likely corresponds to an increase of
>1000% in the enzyme activity in the MC chloroplasts.
Lesson 3: enzyme activities must be properly
regulated
The activity of C4-specific PPDK is strictly regulated by
light in C4 plants through protein phosphorylation by the
PPDK regulatory protein, which catalyses both
Engineering a single-cell C4 pathway into rice | 3025
phosphorylation and dephosphorylation (Burnell and
Hatch, 1985). The activity of maize PPDK in rice leaves is
regulated by light, as it is in C4 leaves, and this is an
indication that the rice PPDK regulatory protein recognizes
and regulates the maize isozyme in the same way as the
corresponding protein does in C4 leaves (Fukayama et al.,
2001; Taniguchi et al., 2008). The activity of the chloroplastic NADP-MDH is also strictly regulated by light in both
C3 and C4 plants through the thioredoxin cascade (Miginiac-Maslow et al., 2000). It was confirmed that the
sorghum MDH overproduced in rice leaves was active only
under illumination, although the activation by light proceeded more slowly than did the activation of endogenous
MDH in non-transgenic rice (Taniguchi et al., 2008).
In contrast, maize PEPC and rice NADP-ME are active
both in the light and in darkness. The activity of plant-type
PEPC is regulated by two different but interactive mechanisms, one that functions through various metabolite
effectors and another that involves reversible phosphorylation of a conserved serine residue at the N terminus (Vidal
and Chollet, 1997). After phosphorylation, PEPC becomes
less sensitive to its feedback inhibitor malate and more
sensitive to the activator glucose 6-phosphate, thereby
attaining higher activity (Vidal and Chollet, 1997). The
activity of maize PEPC in rice leaves is regulated by protein
phosphorylation, but in a manner opposite to that observed
in C4 leaves: it is phosphorylated at night and dephosphorylated during the middle part of the day, in the same way as
the endogenous rice enzyme (Fukayama et al., 2003, 2006).
This indicates that maize PEPC is more active at night than
during the day. For chloroplastic NADP-ME, no particular
regulation mechanisms have been proposed. It is likely that
its activity and the direction of the reaction it catalyses are
regulated by pH, substrate concentrations, and probably by
the Mg2+ concentration in the chloroplast stroma. So far as
the optimum pH is considered (7.8 for the maize nonphotosynthetic isozyme; Saigo et al., 2004), NADP-ME is
slightly more active in the light than in darkness.
It is desirable that the introduced enzymes be active only
under illumination to minimize adverse metabolic effects. In
fact, overproduction of maize PEPC and rice NADP-ME
together leads to stunting of the rice plants (Fig. 3; Taniguchi
et al., 2008). On this basis, it is hypothesized that the
combination of PEPC with NADP-ME catalyses a futile
pathway that wastes metabolic energy. Based on the
phosphorylation pattern of maize PEPC, this pathway could
be more active at night than during the day in rice leaves.
Lesson 4: what is the key to driving the
C4-like pathway in rice MCs?
To produce quadruple transformants that overproduced all
four enzymes (PEPC, PPDK, NADP-MDH, and NADPME), transgenic lines that overproduced PEPC and PPDK
together were first developed by means of conventional
crossing, and then a gene construct for expression of
sorghum NADP-MDH and rice NADP-ME cDNAs was
introduced into the PEPC3PPDK line (Taniguchi et al.,
Fig. 3. Comparison of the shoot dry weights of transgenic rice
plants that overproduce C4 enzymes independently or in combination. Rice plants were grown in a temperature-controlled
greenhouse under natural light conditions for 7 weeks. The
overproduced enzymes were maize PEPC (PE-2), maize PPDK
(PD-332), sorghum NADP-MDH (MD-36), sorghum MDH and rice
NADP-ME together (MD3ME), and maize PEPC and rice NADPME together (PEdME-8) (see Fukayama et al., 2001, 2003;
Taniguchi et al., 2008). NT, non-transgenic rice. Transgenic rice
plants tended to show stunted growth due to somaclonal
variation that occurred during the course of cell cultivation (see
Lesson 5).
2008). The leaf activities of PEPC and PPDK in the
resulting quadruple transformants were therefore the same
as in the PEPC3PPDK line (i.e. 40- to 50-fold and 4- to 5fold the levels in the non-transformants, respectively).
Unexpectedly, four homozygous lines selected from the
quadruple transformants showed similar activity of NADPMDH and NADP-ME, at 7- to 13-fold and 3- to 4-fold the
levels in the non-transformants, respectively. As described
above, the 400% increase in leaf NADP-ME activity
corresponded to an increase to 2000% of the enzyme
activity level in the MC chloroplasts.
These quadruple transformants showed a higher CO2
assimilation rate than that of the original PEPC3PPDK
cross (Taniguchi et al., 2008): the CO2 assimilation rate,
which had been lowered by the action of overproduced
PEPC in the PEPC3PPDK line, was restored to levels
comparable to or higher than those in non-transformants.
Since triple transformants that overproduced three of the
four enzymes (but not NADP-MDH) did not show
enhanced CO2 assimilation, the increased NADP-MDH
activity appears to be the key to the enhanced CO2
assimilation. This finding was unexpected, since there was
no previous report of significant elevation of NADP-MDH
activity as a result of a shift from C3 to C4 photosynthesis in
Hydrilla, and it had been generally believed that the activity
of NADP-MDH in C3 MC chloroplasts was high enough
for operation of the C4-like pathway; the NADP-MDH
activity was an order of magnitude higher than those of the
3026 | Miyao et al.
other three enzymes in the leaves of non-transgenic rice
(Taniguchi et al., 2008). As described in Lesson 2, it is
necessary to shift the equilibrium of the rice C3-specific
NADP-ME towards decarboxylation of malate under
illumination. It is likely that NADP-MDH supports the
decarboxylation reaction of NADP-ME by lowering the
NADPH to (NADP++NADPH) ratio in the stroma and by
facilitating the import of OAA into the chloroplasts.
Further elevation of NADP-MDH activity is expected to
improve CO2 assimilation by the quadruple transformants.
The use of other NADP-ME isozymes with different
kinetic properties might be another choice, but it is important
to couple reactions by NADP-MDH and NADP-ME so as to
avoid raising the stromal NADPH level.
In addition to the importance of increased NADP-MDH
activity, there is no information available on the relative
abundance of introduced enzymes required for operation of
the C4-like pathway. Based on the absolute activities of the
introduced enzymes in leaf extracts from the quadruple
transformants (i.e. 3, 0.1, and 2–3 lmol mg1 protein min1
for PEPC, PPDK, and NADP-MDH, respectively; Taniguchi
et al., 2008), PPDK activity must be further increased to
drive a closed cycle. This cannot be achieved simply by
increasing the PPDK protein level because of limitations on
the activation of the overproduced enzyme by the endogenous PPDK regulatory protein (Fukayama et al., 2001;
Taniguchi et al., 2008). To increase PPDK activity by an
order of magnitude above the present level, both the PPDK
regulatory protein and the PPDK protein itself must be
overproduced in rice leaves. The reduction of PEPC and
NADP-ME activities seems to beneficially adjust the
activity balance between the four enzymes and to mitigate
the loss of metabolic energy through futile pathways in
transgenic rice leaves (Lesson 3).
Other points to be considered are the kinetic properties of
the introduced enzymes and PEP transport activity. At
present, no experimental data are available to enable discussion of these points. A mathematical modelling and systems
biology approach, rather than trial and error experiments,
might be a more productive way to clarify these points.
A peculiar feature that was identified for the first time
was phosphorylation of PEPC at night in rice leaves
(Fukayama et al., 2003). One of three rice PEPC kinases
(OsPPCK3) is responsible for this nocturnal phosphorylation (Fukayama et al., 2006). It had long been considered
that PEPC was phosphorylated only during the daytime in
both C3 and C4 plants. Among the 30 plant species tested,
however, PEPC was phosphorylated during the daytime in
only 12 species (Fukayama et al., 2006). It was later found
that the extent of the phosphorylation at night in rice leaves
depended on the nitrogen source, and became less marked
when rice plants were grown with NH+4 (Fig. 4). Similar
effects of the nitrogen source were reported for PEPC in pea
(Pisum sativum L.) leaves, in which diurnal oscillation of the
PEPC activity (high in the daytime and low at night)
disappeared when plants grown with NO–3 were transferred
into a medium containing NH+4 as the nitrogen source
(Leport et al., 1996). By analysing the expression pattern of
OsPPCK3 in rice plants grown in soil (data not shown), it
was recently realized that a commercial soil mixture used
was rich in NO–3 even when waterlogged. This finding was
surprising, but it is very important for introduction of the
C4-like pathway, since it suggests that the stunting caused
by overproduction of both PEPC and NADP-ME (Lesson
Lesson 5: what has been learned from rice
Rice has been used as a model crop species in plant genomics.
However, it was found that it is unique in its physiology and
primary metabolism among the plant materials generally used
for molecular biology and physiology research. One important difference is that rice plants preferentially use ammonium
(NH+4 ) and thrive better with NH+4 than with nitrate (NO–3)
as their nitrogen source. Many upland crop species, such as
wheat, barley (Hordeum vulgare L.), tobacco (Nicotiana
tabacum L.), tomato (Lycopersicon esculentum L.), and
spinach (Spinacea oleracea L.) preferentially use NO–3. The
waterlogged soil in paddy fields is a reducing environment, so
that nitrogen fertilizers tend to be converted into NH+4 . It is
therefore reasonable that rice would have evolved or been
bred for adaptation to such a habitat by acquiring mechanisms for efficient utilization of NH+4 .
Fig. 4. Effects of the nitrogen source on phosphorylation of PEPC
in rice leaves. Rice plants were grown hydroponically with either 1
mM NO–3 or 1 mM NH+4 as the nitrogen source in a growth
chamber with a day length of 14 h for ;6 weeks. Leaf blades were
harvested from the uppermost fully expanded leaves (the ninth
leaves) at the indicated times of day for these analyses. (A)
Expression of the rice PEPC kinase 3 gene (OsPPCK3) in nontransgenic rice. RT-PCR was carried out as described previously
(Fukayama et al., 2006). (B) Phosphorylation of maize PEPC
in transgenic rice PE-2 (Fukayama et al., 2003). Immunoblot
profiles with antibodies specific to maize C4-specific PEPC in the
phosphorylated form (anti-PEPC-P; Ueno et al., 2000) and specific
to maize C4-specific PEPC in both phosphorylated and dephosphorylated forms (anti-PEPC) are shown. A maize leaf blade
harvested at 13:00 (ML) was loaded as the positive control.
Engineering a single-cell C4 pathway into rice | 3027
3) could be largely mitigated when rice plants are grown
only with NH+4 . The nocturnal phosphorylation of PEPC
was also observed in two hydrophytic weeds that are
commonly found in paddy fields (Fukayama et al., 2006).
It is therefore likely that the nocturnal phosphorylation
plays some role in adaptation to waterlogged soil.
Another recently discovered unique feature is that rice
has PEPC intrinsically located in the chloroplast, Osppc4
(Masumoto et al., 2010). The Osppc4 gene shows a high
expression level in leaf MCs, and the Osppc4 protein
accounts for about one-third of the total PEPC protein in
the leaf blades. Genes encoding the chloroplastic PEPC
have been found in cultivated and wild Oryza species, all of
which are adapted to waterlogged soil. It is suggested that,
in addition to glycolysis, the genus Oryza has a unique
route to provide organic acids for NH+4 assimilation that
involves the chloroplastic PEPC, and that this route is
crucial for growth with NH+4 as the nitrogen source
(Masumoto et al., 2010). The activity of the chloroplastic
PEPC in leaf extracts of non-transgenic rice is between 0.02
and 0.03 lmol mg1 protein min1 (Masumoto et al., 2010).
Although this value is lower than the activities of the
introduced enzymes in the quadruple transformants (see
Lesson 4), the endogenous PEPC inside the chloroplasts
may shortcut the C4-like pathway in rice MCs.
It thus appears that both the carbon and nitrogen
metabolism of rice plants differ from those of upland plant
species. For metabolic engineering to succeed, it will be
necessary to understand these aspects of the basic metabolism of a target plant species. Unfortunately, the primary
metabolism of rice plants has not yet been completely
described.
The last point to be mentioned is technical, and involves
how to minimize genome alterations (somaclonal variation)
that can occur during the course of cell cultivation and the
regeneration of plants from the cultured cells (Labra et al.,
2001). It appears that somaclonal variation is inevitable
when calli are used for transformation. Unfortunately, all
procedures for rice transformation that are presently available use calli induced from mature seeds (Hiei et al., 1994;
Toki et al., 2006), and methods similar to the floral dip
transformation that is used for Arabidopsis are unavailable.
To minimize genome alterations, researchers should avoid
prolonged cell cultivation to prevent accumulation of
undesirable phenotypes, such as stunting and morphological abnormalities, in multiple transformants. Finding an
alternative to rice callus transformation would also represent a significant breakthrough in this context.
ME reaction. The importance of NADP-MDH was also
recognized only after the initial series of experiments had
been completed. This is not the only case in which
transgenic plants have shown unexpected phenotypes,
leading to the discovery of novel regulatory mechanisms
and metabolic interactions that would not have been
discovered using traditional physiological and biochemical
approaches. The transgenic approach is therefore a powerful
tool for basic research in plant science. These attempts to
introduce the C4-like pathway in rice have also deepened
the understanding of the physiology of this species. There is
little doubt that such an approach will contribute greatly in
future towards the metabolic engineering of rice plants.
For engineering a single-cell C4-like pathway into rice,
the presence of the endogenous PEPC inside the MC
chloroplasts has emerged as a novel problem that must be
addressed. At the same time, strategies to improve the
quadruple transformants have been revealed by this research. Among them, further elevation of NADP-MDH
activity seems to be the most promising approach. To
answer the question of whether a single-cell C4-like pathway
can improve the photosynthetic performance of rice,
additional step by step research informed by the lessons
described in this article will be required.
Acknowledgements
The authors are grateful to Professor Osamu Ueno, Kyushu
University, Japan, for performing the immunoelectron
microscopy analysis. This work was supported in part by
a grant from the Ministry of Agriculture, Forestry, and
Fisheries of Japan (Genomics for Agricultural Innovation,
GPN00006) to M.M.
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