Journal of Experimental Botany, Vol. 59, No. 7, pp. 1799–1809, 2008
doi:10.1093/jxb/ern016 Advance Access publication 2 March, 2008
SPECIAL ISSUE RESEARCH PAPER
Overproduction of C4 photosynthetic enzymes in transgenic
rice plants: an approach to introduce the C4-like
photosynthetic pathway into rice
Yojiro Taniguchi1, Hiroshi Ohkawa1,*, Chisato Masumoto1, Takuya Fukuda1, Tesshu Tamai1,†,
Kwanghong Lee1,‡, Sizue Sudoh1, Hiroko Tsuchida1, Haruto Sasaki2, Hiroshi Fukayama1,§ and
Mitsue Miyao1,{
1
Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences (NIAS),
Kannondai, Tsukuba 305-8602, Japan
2
University Farm, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Midoricho,
Nishitokyo, Tokyo 188-0002, Japan
Received 15 October 2007; Revised 22 December 2007; Accepted 11 January 2008
Abstract
Four enzymes, namely, the maize C4-specific phosphoenolpyruvate carboxylase (PEPC), the maize C4specific pyruvate, orthophosphate dikinase (PPDK),
the sorghum NADP-malate dehydrogenase (MDH), and
the rice C3-specific NADP-malic enzyme (ME), were
overproduced in the mesophyll cells of rice plants
independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate
of photosynthetic CO2 assimilation, while in the case
of PEPC it was slightly reduced. The reduction in CO2
assimilation in PEPC overproduction lines remained
unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by
the combined overproduction of PPDK, ME, and MDH
to reach levels comparable to or slightly higher than
that of non-transgenic rice. The extent of the restoration of CO2 assimilation, however, was more marked at
higher CO2 concentrations, an indication that overproduction of the four enzymes in combination did not
act to concentrate CO2 inside the chloroplast. Transgenic rice plants overproducing the four enzymes
showed slight stunting. Comparison of transformants
overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME
was responsible for stunting, and that overproduction
of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as
possibilities and limitations of introducing the C4-like
photosynthetic pathway into C3 plants, are discussed.
Key words: C4 photosynthesis, malate dehydrogenase, malic
enzyme, overproduction, phosphoenolpyruvate carboxylase,
pyruvate, orthophosphate dikinase, transgenic rice.
Introduction
Most terrestrial plants, including many important crops
such as rice (Oryza sativa), wheat (Triticum aestivum),
soybean (Glycine max), and potato (Solanum tuberosum)
assimilate CO2 through the C3 photosynthetic pathway
(the Calvin cycle), and are classified as C3 plants.
However, some plants, such as maize (Zea mays) and
sugarcane (Saccharum officinarum), possess the C4 photosynthetic pathway in addition to the C3 pathway, and
these are classified as C4 plants. The C4 pathway acts to
* Present address: Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
y
Present address: Chiben-Gakuen High School, Gojo, Nara 637-0042, Japan.
z
Present address: Plant Pathology Department, University of Nebraska-Lincoln, Lincoln, NE 68583-0722, USA.
§
Present address: Graduate School of Agriculture, Kobe University, Kobe 657-8501, Japan.
{
To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: Ca, the ambient CO2 concentration; Ci, the intercellular CO2 concentration; d13C, the relative content of 13C in total carbon atoms; MDH,
malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate,
orthophosphate dikinase; PPFD, photosynthetically active photon flux density; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; SD, standard
deviation; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1800 Taniguchi et al.
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 high photosynthetic capacity and higher water and
nitrogen use efficiencies (Hatch, 1987). Consequently, the
transfer of C4 traits to C3 plants is one strategy being
adopted for improving the photosynthetic performance of
C3 plants.
The C4 pathway consists of three key steps: (i) the
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 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 initial carboxylation. This
separation occurs through the bundle sheath and mesophyll cells in typical/classical C4 plants (Hatch, 1987;
Leegood, 2002), and through two distant subcellular
compartments in the recently discovered single-cell C4
plants (Edwards et al., 2004). Structural and biochemical
features of chloroplasts in these two sites are also
different. It has been proposed that such compartmentation and chloroplast differentiation, together with structural adaptation to minimize CO2 diffusion from the CO2
release/elevation site, are essential for C4 photosynthesis.
By contrast, in some aquatic plants C4 photosynthesis is
accomplished in a single cell without any compartmentation and chloroplast differentiation (Bowes et al., 2002;
Leegood, 2002). At least three submerged macroalga, all
of which are freshwater monocots, perform such C4
photosynthesis (Bowes et al., 2002). Hydrilla verticillata
has been the best documented. It is a facultative C4 plant
that shifts from C3 to C4 photosynthesis under low CO2
conditions without any structural modifications of leaf
cells. The C4-like pathway of Hydrilla is relatively simple
(Fig. 1). Upon the shift to C4 photosynthesis, genes
encoding the C4-specific isoforms of PEPC, PPDK, and
NADP-malic enzyme (ME) are up-regulated (Rao et al.,
2002, 2006; Estavillo et al., 2007). Although the shift is
accompanied by the up-regulation of various genes (Rao
et al., 2006), protein components required for the
operation of the C4-like pathway remain to be solved.
The recent application of recombinant DNA technology
has made considerable progress in molecular engineering
of photosynthetic genes. It enabled us to overproduce
enzymes involved in the C4 pathway at high levels and in
desired locations in the leaves of C3 plants (for a review,
see Matsuoka et al., 2001; Miyao, 2003), and a variety of
transgenic C3 plants overproducing C4 enzymes have been
generated and intensively analysed (for a review, see
Häusler et al., 2002; Miyao, 2003; Miyao and Fukayama,
2003). We are attempting to introduce the simplest model
Fig. 1. Introduction of the C4-like photosynthetic pathway of Hydrilla
verticillata into the mesophyll cell of C3 plants. It has been proposed
that, if PEPC, PPDK, and NADP- ME can be overproduced, the C4-like
pathway of Hydrilla might operate in the mesophyll cell of C3 plants.
The activity of NADP-MDH in C3 mesophyll cells is high, and it is
considered that activation of the endogenous enzyme may be sufficient
for operation of the pathway. OAA, oxaloacetate; Mal, malate; PEP,
phosphoenolpyruvate; Pyr, pyruvate.
of C4 photosynthesis, the C4-like pathway of Hydrilla,
into the mesophyll cell of a C3 plant, rice (Fig. 1). Until
now, we have produced more than ten sets of different
transgenic rice overproducing four C4 enzymes, namely,
PEPC, PPDK, NADP-ME, and NADP-malate dehydrogenase (MDH), independently or in combination. In this
study, photosynthesis and growth of these transgenic rice
plants were analysed and compared. It was found that
overproduction of all four enzymes in combination
slightly improved photosynthesis, but at the same time
caused slight but reproducible stunting of the transgenic
plants.
Materials and methods
Plant materials
For the production of transgenic rice plants overproducing a single
enzyme in the mesophyll cell of rice plants, five different gene
constructs were used. They contained the intact maize C4-specific
gene for overproduction of PEPC and PPDK (Ku et al., 1999;
Fukayama et al., 2001) or the full-length cDNA fused to the rice
Cab promoter (Sakamoto et al., 1991) for overproduction of the
maize C4-specific NADP-ME, the rice C3-specific isoform (Tsuchida et al., 2001), and the sorghum NADP-MDH. The cDNA
clone for the sorghum MDH (accession No. X53453) was
a generous gift from Professor M Miginiac-Maslow, CNRS/
Université Paris-Sud, France. The constructs described above were
cloned into a binary vector pIG121Hm containing a hygromycin
resistance gene, and introduced into rice plants (cv. Kitaake) via
Agrobacterium-mediated gene transfer. Transgenic lines in which
the T1 generation exhibited a segregation ratio of around 1:3
(assessed by hygromycin resistance) were chosen for further
analysis, and levels of the introduced enzyme protein in the leaves
were screened by SDS-PAGE. Protein levels were determined from
intensities of protein bands after staining with Coomassie Brilliant
Blue R-250 for PEPC and PPDK, or from those after immunoblotting for ME and MDH. Plants with the highest protein level were
taken as homozygous and self pollinated. Homogeneities of the
level of the introduced enzyme were confirmed in the T2 and T3
generations.
Overproduction of C4 enzymes in rice leaves 1801
Enzyme activities were determined spectrophotometrically at 30
C. The PEPC activity was assayed by the method of Fukayama
et al. (2003). The activity assayed at pH 7.5 in the presence of 5
mM glucose-6-phosphate was taken as the maximum activity. The
PPDK activity was assayed after activation of the enzyme by an
incubation of the protein extract at 25 C for 1 h (Fukayama et al.,
2001). The NADP-ME activity was assayed after desalting the
protein extract through a gel filtration column as described
previously (protocol 2, Tsuchida et al., 2001).
For NADP-MDH, extraction of leaf soluble protein and the
activity assay were performed by the method of Tsuchida et al.
(2001). The activity measured immediately after protein extraction
was taken as the in vivo activity. The maximal potential activity was
measured after incubation under a nitrogen gas stream of the protein
extract with 1 mM dithiothreitol and 5 lM recombinant thioredoxin
m (a generous gift from Professor T Hisabori and Dr K Motohashi,
Tokyo Institute of Technology, Japan) at 25 C for 40 min.
Transgenic rice plants overproducing more than one enzyme
were produced by crossing two different single transformants and/or
the second gene introduction into transformants. Gene constructs
used for the second gene introduction contained the full-length
cDNA for the rice C3-specific ME fused to the rice Cab promoter
for overproduction of ME alone, or the full-length cDNAs for the
rice C3-specific ME and the sorghum MDH, each of which were
fused to the rice Cab promoter, for overproduction of MDH and
ME together. They were cloned into a binary vector pGTV35Sbar
containing the bialaphos resistance gene (a generous gift from Dr F
Takaiwa, National Institute of Agrobiological Sciences, Japan) and
introduced into rice plants. Homozygous lines with a single insertion site were selected by the segregation ratio (assessed by
bialaphos resistance) and the protein level as described above.
Transgenic rice plants used in this study are listed in Table 1.
Maize and sorghum plants were planted in vermiculite. Unless
otherwise stated, rice plants were planted in 1/10 000- or 1/5000-are
Wagner pots containing 1.0 l or 3.0 l of commercial soil mixture
(Bonsol No. 1, Sumitomo Chemicals, Japan). They were grown
under natural light conditions in temperature-controlled greenhouses
at day and night temperatures of 26 C and 21 C, respectively.
When indicated, plants were grown in a growth chamber at 26/21
C day/night cycle with a day period for 14 h under illumination
with white light at a photosynthetically active photon flux density
(PPFD) of 500–600 lmol m2 s1.
Analytical procedures
Protein determination and SDS–PAGE were carried out as described previously (Tsuchida et al., 2001). After SDS–PAGE, the
gel was stained with Coomassie Brilliant Blue R-250 or subjected
to immunoblotting using antisera raised against the sorghum MDH
(a generous gift from Professor M Miginiac-Maslow), the maize C4specific PEPC, the phosphorylated form of the maize C4-specific
PEPC (a generous gift from Professor K Izui, Kinki University,
Japan) (Ueno et al., 2000), or the recombinant rice C3-specific ME
produced in Escherichia coli. Immunoreacted bands were visualized
by the alkaline phosphatase reaction (Bio-Rad).
Gas exchange of the uppermost fully expanded leaf was
measured with an open gas-exchange system (LI-6400, Li-Cor,
NB, USA) as described previously (Fukayama et al., 2003).
Measurements were performed at a leaf temperature of 25 C, 21%
Extraction of leaf soluble protein and enzyme assays
Segments of about 3 cm were harvested from the mid-section of the
uppermost fully expanded leaves at 11.00–12.00 h and immediately
frozen in liquid nitrogen until use. Total leaf soluble protein extracts
were prepared as described previously (Tsuchida et al., 2001). For
assay of the PPDK activity, 5 mM pyruvate and 2 mM KH2PO4
were included in the extraction buffer (Fukayama et al., 2001).
Table 1. List of transgenic rice plants used in this study
Introduced enzymes were the maize C4-specific PEPC, the maize C4-specific PPDK, the sorghum NADP-MDH, and the rice C3-specific NADP-ME.
The maximum activities of introduced enzymes in the leaves are expressed as fold increase over those of non-transgenic rice plants. In some
transgenic lines, PPDK could not be fully activated both in vivo (see Fig. 4) and in vitro. PPDK activities from leaf samples harvested at 11.00–12.00
h are presented for these lines. Absolute activities in non-transgenic rice were 0.07, 0.02, 0.25, and 0.01 lmol mg1 protein min1 for PEPC, PPDK,
NADP-MDH, and NADP-ME, respectively.
Plant name
Single transformant
PE-84
PE-2
PD-332
PD-259
PD-317
MD-36
ME-4
Double transformant
PE3PD
MD3ME
PEdME-104
Triple transformant
Trip-73
Quadruple transformant
Quad-3
Quad-11
Quad-15
Quad-98
Generation
Relative activity
Production procedure
PEPC
PPDK
MDH
ME
T5, homo
T>5, homo
T>5, homo
T5, homo
T5, homo
T4, homo
T2, homo
20
40–50
–
–
–
–
–
–
–
–
–
–
–
–
40
–
–
–
–
–
–
–
6
F>4, homo
F2, hetero
T1, hetero
T2, homo
40–50
–
40–50
40–50
4–5
–
20/40
–
–
–
3/6
3/6
6
Crossing (PE-23PD-332)
Crossing (MD-363ME-4)
Gene introduction to PE-2
T2, T3, homo
40–50
4–5
–
3
Gene introduction to PE3PD
T2, homo
40–50
40–50
40–50
40–50
4–5
4–5
4–5
4–5
10–11
12–13
7–8
10–11
3
4
3
3
Gene introduction to PE3PD
4–5
10–12
18–20
–
–
–
–
–
Gene introduction
Gene introduction
Gene introduction
Gene introduction
1802 Taniguchi et al.
O2, a PPFD of 1200 lmol m2 s1, and VPD of 1.0–1.2 kPa. The
Ci response curve of CO2 assimilation was obtained with a single
leaf by decreasing the ambient CO2 concentration (Ca) stepwise
from 880 to 45 ll l1.
For determination of d13C, three seedlings of rice plants were
transplanted in a plastic box (15.5 cm length36 cm width310 cm
depth) containing 0.6 l of the soil mixture, and grown in the
greenhouses. Middle portions of the leaf blade (about two-thirds in
length) were harvested from the uppermost fully expanded leaf (7th
leaf) at 13.00–14.00 h on sunny days, immediately frozen in liquid
nitrogen, and dried at 80 C for 2 d. The dried sample was ground
to a fine powder using a mortar and pestle, and further dried at 80
C for 1 d. The 13C content was determined using an elemental
analyser (NC2500, Thermoquest, San Jose, CA, USA) and a mass
spectrometer (Delta Plus System, Thermoquest) (Sasaki et al.,
2005), and the d13C value relative to Pee Dee Belemnite was
calculated using a 13CO2/12CO2 gas mixture and flour powder of
known d13C values as standards.
Results
Effects of overproduction of a single C4 enzyme on
photosynthesis and growth of rice plants
In the leaves of C3 plants, PEPC has an anaplerotic role
replenishing the tricarboxylic acid cycle with intermediates, which are withdrawn for nitrate assimilation and the
subsequent amino acid synthesis (Melzer and O’Leary,
1987). In accordance with this function, it has previously
been observed that overproduction of the maize C4specific PEPC in rice leaves enhanced respiration in the
light (Fukayama et al., 2003). At the same time, the
Rubisco activity estimated from the slope of the Ci
response curve of CO2 assimilation was reduced
(Fukayama et al., 2003). As a result, transgenic rice plants
overproducing PEPC showed slightly suppressed CO2
assimilation. Figure 2A compares Ci response curves of
transgenic rice plants overproducing different levels of the
maize PEPC. It is clearly seen that the suppression was
more marked in the transformant with a higher level of
PEPC. Elevation of the PEPC activity up to 50-fold over
that of non-transgenic rice did not significantly affect the
growth of rice plants, while further elevation caused slight
stunting. It was reported that overproduction of PEPC
enhanced stomatal opening, thereby improving CO2
assimilation of rice plants (Ku et al., 2000). Stomatal
responses remained unaffected in our transgenic rice
plants overproducing PEPC (Fukayama et al., 2003).
Overproduction of the maize C4-specific PPDK did not
affect CO2 assimilation and growth of rice plants at all,
unless the foreign PPDK accumulated above some
threshold level (Fukayama et al., 2001). As shown in Fig.
2B, the Ci response curve of the homozygous line PD-332
completely matched with that of non-transgenic rice. The
other line PD-259 with a higher PPDK level showed
slightly suppressed CO2 assimilation. This effect probably
resulted from nitrogen deficiency due to a high nitrogen
demand of foreign PPDK as discussed previously
Fig. 2. Dependences on Ci of CO2 assimilation rate of transgenic rice
plants overproducing PEPC and PPDK. (A) Transgenic rice overproducing PEPC (PE-84 and PE-2). (B) Transgenic rice overproducing
PPDK (PD-332 and PD-259). Results from non-transgenic rice (NT;
crosses) and the hybrid between PE-2 and PD-332 (PE3PD; small filled
circles) are also shown. Two different plants from each line were
analysed. CO2 assimilation rates were measured at the same Ca values
from 45 to 880 ll l1.
(Fukayama et al., 2001). Overproduction of PPDK in
addition to PEPC did not affect CO2 assimilation, and the
hybrid between PE-2 and PD-332 showed the same Ci
response curve as PE-2 (Fig. 2A). A previous study
demonstrated that transgenic tobacco plants overproducing the plastidic PPDK from the facultative Crassulacean
acid metabolism (CAM) plant Mesembyanthemum crystallinum produced more seeds per seed capsule and
heavier seed capsules than non-transgenic plants, and
proposed that this effect was ascribable to overproduction
of PPDK in seeds (Sheriff et al., 1998). Although the
maize PPDK was overproduced in spikelets of our
transgenic rice plants (Fukayama et al., 2001), increased
grain yield was not always observed.
As demonstrated previously (Tsuchida et al., 2001),
overproduction of the maize C4-specific NADP-ME led to
enhanced photoinhibition of photosynthesis, leaf chlorophyll bleaching and serious stunting, even when the
activity of the maize enzyme was only several fold that of
non-transgenic rice. These detrimental effects resulted
from an increase in the NADPH/NADP+ ratio in the
chloroplast stroma and suppression of photorespiration by
depletion of malate, which is exported from the stroma in
exchange with 2-oxoglutarate for photorespiration (Tsuchida et al., 2001). Overproduction of the rice C3-specific
isoform did not affect photosynthesis and growth of rice
plants (data not shown). Overproduction of the sorghum
NADP-MDH up to 40-fold wild-type activities had little
effect on plant growth (see Fig. 6A).
Activity regulation of foreign C4 enzymes in rice leaves
The activity of plant-type PEPC is regulated by two
different, but interactive mechanisms; one is through
Overproduction of C4 enzymes in rice leaves 1803
various metabolite effectors, and the other is reversible
phosphorylation of a conserved serine residue near the N
terminus (Vidal and Chollet, 1997). Upon phosphorylation PEPC becomes less sensitive to its feedback inhibitor
malate and more sensitive to the activator glucose6-phosphate, thereby attaining a higher activity (Vidal
and Chollet, 1997). The maize C4-specific PEPC overproduced in transgenic rice leaves undergoes activity
regulation by protein phosphorylation but in a manner
opposite to that observed in the leaves of C4 plants: it is
phosphorylated at night and dephosphorylated during the
middle part of the day, in just the same way as the rice
endogenous enzyme (Fukayama et al., 2001, 2006). One
of three rice PEPC kinase genes is responsible for this
nocturnal phosphorylation (Fukayama et al., 2006). As
shown in Fig. 3, the phosphorylation pattern of the maize
PEPC in the transgenic rice leaves remained unaffected by
additional overproduction of PPDK alone in PE3PD or
in combination with ME and MDH in the quadruple transformants, and the maize PEPC was phosphorylated at
night in all cases. The level of phosphorylated PEPC at
night was slightly higher in Quad-3 and Quad-11 than in
PE-2 and PE3PD.
The activity of the C4-specific PPDK is strictly
regulated by light in C4 plants through protein phosphorylation by the PPDK regulator protein, which catalyses
both phosphorylation and dephosphorylation (Burnell and
Hatch, 1985). As demonstrated previously (Fukayama
et al., 2001), the maize PPDK expressed in transgenic
rice leaves undergoes activity regulation by light as in
the leaves of C4 plants (Fig. 4). In maize, transgenic rice
plants overproducing PPDK, and non-transgenic rice,
PPDK was activated at the onset of illumination and deactivated in the dark. In transgenic rice leaves expressing
the maize PPDK at very high levels, PPDK failed to be
fully activated even after 14 h illumination. The maize
PPDK in protein extracts from transgenic rice leaves was
activated in vitro by incubation with phosphate and
inactivated by ADP, as observed in maize (data not
shown).
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).
The sorghum MDH expressed in rice leaves also underwent activity regulation by light. The rate of activation
by light in transgenic rice, however, was lower than in C4
plants and non-transgenic rice. In sorghum and nontransgenic rice, the MDH activity reached steady-state
within 2 min after the onset of illumination at a PPFD of
1000 lmol m2 s1, while it took about 60 min in the
homozygous line MD-36, in which maximum MDH
activity was 40-fold that of non-transgenic rice (data not
shown). The steady-state level of activation of the enzyme
was the same in MD-36 and non-transgenic rice, and it
was about 50% under full sunlight.
Fig. 3. Phosphorylation of the maize PEPC in the leaves. Leaf samples
were harvested at 12.00 h (L) and 24.00 h (D). Immunoblot profiles
with antibodies specific to the maize C4-specific PEPC in the
phosphorylated form (anti-PEPC-P) (Ueno et al., 2000) and to the
maize C4-specific PEPC in both phosphorylated and unphosphorylated
forms (anti-PEPC) are shown. PE-2, transgenic rice overproducing
PEPC; PE3PD, transgenic rice overproducing PEPC and PPDK (PE23PD-332 hybrid); Quad-3 and Quad-11, transgenic rice overproducing
PEPC, PPDK, MDH, and ME, which had been produced from PE23PD-332 (see Table 1).
Fig. 4. Light-dependent regulation of PPDK activity in the leaves.
Maize and rice plants were grown in a growth chamber on the day/night
cycle for 5 weeks. Leaf samples were harvested at designated times.
The PPDK activity was assayed immediately after extraction of leaf
soluble protein. PD-259 and PD-317, transgenic rice overproducing
PPDK. Quad-3, transgenic rice overproducing PEPC, PPDK, MDH, and
ME. The levels of PPDK protein were 6% and 21% of total leaf soluble
protein for PD-259 and PD-317, respectively, and 2% for Quad-3. NT
stands for non-transgenic rice, and L and D indicate light and dark
periods, respectively.
Production of transgenic rice plants overproducing
more than one enzyme
As described above, overproduction of the maize C4specific ME in rice leaves led to serious stunting of plants.
Such detrimental effects of the maize enzyme were not
mitigated by additional overproduction of the sorghum
MDH (Fig. 5A), which takes part in the export of
reducing equivalents from the chloroplast (MiginiacMaslow et al., 2000). Overproduction of all three enzymes
1804 Taniguchi et al.
protein level exceeded 6% of total leaf soluble protein,
the growth of calli in culture was seriously inhibited and,
as a result, the efficiency of gene introduction became
one-order of magnitude lower than that of non-transgenic
rice. Therefore, in all experiments involving double
transformation, PD-332 was used as the transgenic explant
source, in which PPDK level was around 2% of total
protein, although the transformation efficiency was still
reduced. Overproduction of PEPC did not have such an
effect.
Two sets of double transformants were first produced by
crossing: the PE3PD cross using lines PE-2 and PD-332
as parents, and the MD3ME cross using MD-36 and ME4 as parents (Table 1). Another double transformant
overproducing PEPC and ME (PE ME) were produced by
the introduction of the rice ME gene construct into PE-2.
To produce triple and quadruple transformants, the double
transformant PE3PD was transformed with the rice ME
gene construct and the construct containing the rice ME
and sorghum MDH genes in tandem, respectively. All
these multiple transformants set seeds normally and their
fertile progeny were successfully generated.
d
Fig. 5. Stunting of rice plants caused by overproduction of the maize
C4-speific NADP-ME. Non-transgenic rice (A) and the double transformant PE3PD (PE-23PD-332; B) were introduced with the tandem
gene construct containing the maize C4-specific NADP-ME cDNA and
the sorghum NADP-MDH cDNA. Primary transformants grown under
weak light illumination are shown. The levels of the maize ME in
transgenic rice leaves were monitored by immunoblotting.
together, namely, PEPC, PPDK, and MDH, was not
effective in reversing this effect either (Fig. 5B). Therefore, it was subsequently chosen to use the rice C3-specific
ME for transformation instead of the maize enzyme. Thus,
enzymes introduced in the multiple enzyme transgenics
were the maize C4-specific PEPC, the maize C4-specific
PPDK, the sorghum MDH, and the rice C3-specific ME,
the latter three of which are targeted to the chloroplast
(Fig. 1). Multiple enzyme transformants were produced by
either crossing two different single enzyme transformants
or by the introduction of the second gene construct into
transformants, or using a combination of both approaches.
All transformants used in this study had a single transgene
per haploid.
During the course of experiments, it was found that very
high levels of foreign PPDK expression hampered gene
introduction. In transgenic lines in which the PPDK
Physiological characteristics of multiple transformants
Although overproduction of any of the four enzymes did
not affect the growth of rice plants, some lines containing
multiple enzymes exhibited stunting. Figure 6 compares
culm length and panicle weight after grain filling,
parameters commonly used for phenotyping the growth
of rice plants. Overproduction of MDH and ME did not
significantly affect culm length or total panicle weight. By
contrast, overproduction of PEPC with PPDK led to slight
decreases in these two parameters. Significant effects on
growth were observed in transformants overproducing
PEPC with ME, namely, the double transformant PEdME104, the triple transformant Trip-73, and the quadruple
transformants Quad-3 and Quad-11. In these transformants, both culm length and total panicle weight were
smaller than those of their respective control transformants. It is noted that line Quad-11 showed occasional
stunting, probably due to some genome alteration caused
by cell cultivation, and therefore, its growth parameters
tended to be underestimated. The stunting caused by
overproduction of PEPC and ME together was more
marked during the vegetative growth stage, and overproduction of MDH had some mitigating effects against
the stunting (data not shown).
Figure 7 compares the response of CO2 assimilation rate
to Ci in transformants containing multiple C4 enzymes.
Overproduction of MDH with ME had no detectable
effect on CO2 assimilation, and the Ci response curve of
the MD3ME cross was almost identical to that of nontransgenic rice plants. As described above, PE-2 overproducing PEPC showed slightly lower CO2 assimilation
Overproduction of C4 enzymes in rice leaves 1805
Fig. 6. Growth of multiple transgenic rice plants after grain filling. Culm length (upper) and total panicle weight (lower) are shown. Multiple
transformants were grown together with non-transgenic rice (NT; open bars) and control transformants (filled bars) in a greenhouse under natural
light conditions. (A) MD3ME overproducing MDH and ME (F2). (B) PEdME-104 overproducing PEPC and ME (T1). (C) triple transformant Trip73 overproducing PEPC, PPDK, and ME. (D) quadruple transformants Quad-3 and Quad-11 overproducing PEPC, PPDK, MDH, and ME. MD3ME
and PEdME-104 lines used were heterozygous, and plants that overproduced introduced enzyme(s) were subjected to analyses. Four different sets of
plants were planted at different times from April to July. Data represent averages 6SD of five plants. Significant differences from non-transgenic rice
according to the Fisher’s LSD test: *P <0.05, **P <0.01.
rates than non-transformants (Fig. 2A). Neither overproduction of ME in addition to PEPC in PE ME-104 nor
overproduction of PPDK and ME in addition to PEPC
(Trip-73) substantially affected CO2 assimilation.
In contrast to the triple transformants, the quadruple
transformants, also overproducing MDH, showed slightly
enhanced CO2 assimilation especially at high Ci values
(Fig. 7D). The CO2 assimilation rate of Quad-3 was
higher than that of the control transformants and, in some
plants, it was comparable to or slightly higher than that of
non-transformants. Quad-11, a line that showed stunting,
also exhibited higher rates of CO2 assimilation than the
control transformants. The same results were reproducibly
obtained when the pot size was large enough: the
enhanced CO2 assimilation was always observed in plants
grown in large pots with 3.0 l soil but not in small pots
with 1.0 l soil (data not shown). The suppression of CO2
assimilation in PE-2 and PE3PD was also influenced by
the pot size, being more marked in larger pots.
Carbon isotope composition is often used as a simple
test for determining the photosynthetic type, since
Rubisco largely discriminates against 13CO2 and C3 plants
show much lower d13C values than C4 plants (Farquhar
et al., 1989). Figure 8 compares d13C values of leaves of
the quadruple transformants. Plants used for these experid
ments were grown in small plastic boxes placed side by
side so that all the plants were exposed to the same air
stream conditions in closed greenhouses. Although differences were small, the d13C value was decreased by
overproduction of PEPC and PPDK together, and increased by overproduction of all four enzymes. These
changes did not result from altered stomatal conductance,
since the Ci/Ca ratio remained unaffected by overproduction of these enzymes (Fig. 7D). It is presumed that
they reflected some alteration of carbon metabolism in
transgenic rice leaves.
Discussion
To determine the effects of overproduction of an enzyme
in transgenic plants accurately, it is necessary to examine
the correlation of the phenotype of transgenic plants with
transgene expression level, preferably at the protein and/or
enzyme activity level. Transgenic rice plants with different levels of the introduced enzyme were compared and it
was concluded that overproduction of a single C4 enzyme
did not improve photosynthesis of rice. Rather, overproduction of the maize PEPC slightly inhibited photosynthesis (Fig. 2A) through stimulation of respiration in
the light and reduction of the Rubisco activity (Fukayama
1806 Taniguchi et al.
Fig. 7. Dependences on Ci of CO2 assimilation rate of multiple transgenic rice plants. (A) MD3ME overproducing MDH and ME (F2,
heterozygous). Relative activities of MDH and ME are indicated inside the figure. (B) PEdME-104 overproducing PEPC and ME (T3). (C) Trip-73
overproducing PEPC, PPDK, and ME. (D, E) Quad-3 and Quad-11 overproducing PEPC, PPDK, MDH, and ME. (E) shows data at a low Ci range in
(D) on an expanded abscissa scale. Results from non-transgenic rice (NT; crosses) and control transformants (small filled circles) are also shown.
Each panel shows results from a set of plants, of which CO2 assimilation rates were measured at the same Ca values from 45 to 880 ll l1.
et al., 2003). The latter effect might be due to enhanced
nitrogen assimilation, which competes with the Calvin
cycle for ATP and reducing power. It has been demonstrated in transgenic potato plants that overproduction of
an engineered unregulated PEPC doubled the rate of dark
respiration but did not substantially affect photosynthetic
characteristics (Rademacher et al., 2002). Some groups
have claimed that overproduction of the maize PEPC
enhanced photosynthesis and increased crop yields of
transgenic rice plants (Jiao et al., 2002; Bandyopadhyay
et al., 2007). However, in these studies, only one or two
lines were examined, and the correlation between the level
of PEPC protein and the extent of observed phenotypes
was not well established.
Of the combinations of C4 enzymes examined, only
overproduction of all the four enzymes had some positive
effects on photosynthesis, and only to a limited extent
(Fig. 7). The reduction in CO2 assimilation rate by
overproduction of PEPC was recovered by overproduction
of all three other enzymes and, in some cases, CO2
assimilation rates reached levels higher than those of nontransgenic rice plants (Fig. 7D). Since overproduction of
PEPC, PPDK, and ME together did not have such effects
(Fig. 7C), the elevation of the MDH activity must be
essential for the restoration/enhancement of photosynthe-
sis. We consider that overproduction of MDH is necessary
for direct uptake of OAA into the chloroplast and also for
the decarboxylation reaction of overproduced ME.
As reported previously, the maize C4-specific ME
overproduced in rice leaves raises the NADPH/NADP+
ratio of the stroma and thereby makes photosynthesis
susceptible to photoinhibition (Tsuchida et al., 2001).
These findings indicate that the reaction of the maize
enzyme is directed toward decarboxylation of malate in
rice leaves. This would not be the case for the rice C3specific isoform even when PEPC was overproduced
together. As shown in Fig. 7, CO2 assimilation was not at
all affected in PE ME-104 and Trip-73 in which ME was
overproduced with PEPC but without MDH, or no
symptom of photoinhibition of photosynthesis was observed in these transgenics. As compared with the C4specific ME, the affinity for NADP+ is one-order of
magnitude lower in the C3-specific isoform (Saigo et al.,
2004). It is likely that the reaction of overproduced rice
enzyme attains equilibrium in the chloroplast under
illumination where the NADPH/NADP+ ratio is always
high, proceeding towards either decarboxylation or carboxylation direction in response to the NADPH/NADP+
status. When ME is overproduced with MDH, NADPH
produced by the decarboxylation reaction of ME can be
d
Overproduction of C4 enzymes in rice leaves 1807
Fig. 8. d13C values of leaves of the quadruple transgenic rice plants.
Four homozygous lines of T2 generation were used. NT and ED stand
for non-transgenic rice and the control transformants PE3PD, respectively. Two panels show the same data on different ordinate scales.
Bidirectional arrows on the right side of the upper panel indicate d13C
values typical for C3 and C4 plants. Averages 6SD of 5–6 plants are
shown. Significant differences from PE3PD according to the Fisher’s
LSD test: *P <0.05, **P <0.01.
consumed by MDH, leaving the level of NADP+ unchanged. At the same time MDH promotes import of OAA
from the cytosol, supporting direct uptake of the product of
overproduced PEPC into the chloroplast. Overproduction
of PPDK would also act to support the decarboxylation
reaction by ME by reducing the level of pyruvate.
The detrimental effects of C4-specific ME have previously been explained by its high Vm and low Km for
malate, which are one-order of magnitude different from
the C3-specific isoform (Tsuchida et al., 2001). Kinetic
parameters for the C3-specific isoform reported previously
have been revised by recent studies using recombinant
proteins, which reported that Km for NADP+ differed
significantly but either kcat NADPþ or Km for malate did not
between two isoforms of maize (Saigo et al., 2004).
It is currently unknown if the C4-like pathway operates
in the quadruple transformants. The d13C value of rice
leaves was decreased by overproduction of PEPC and
PPDK together and increased by overproduction of all
four enzymes, as was the CO2 assimilation rate (Fig. 8).
Even if the C4-like pathway operates in these plants, it
does not seem to act as a CO2 pump in the same way as in
C4 plants. As seen in Fig. 7D and E, the increase in the
CO2 assimilation rate in the quadruple transformants was
larger at higher Ci values, and the CO2 compensation
point was not significantly altered. At low Ci where
photorespiration predominates over CO2 assimilation,
a subtle increase of the CO2 concentration around Rubisco
(Cc) could not significantly affect the CO2 assimilation
rate. At high Ci, in contrast, photorespiration is largely
suppressed and Cc becomes lower than Ci because of
active carboxylation by Rubisco (see Evans and von
Caemmerer, 1996). Under these conditions, some increase
in Cc would be effective in enhancing CO2 assimilation.
For the proper functioning of the C4-like pathway it is
important that overproduced enzymes constitute a closed
cycle so as not to perturb metabolisms of the host plant.
Limited export of PEP from the chloroplast, usually found
in the leaves of C3 plants, may limit the reaction of PEPC
and thereby hamper operation of the cycle.
The present study revealed that overproduction of PEPC
and ME together led to the stunting of transgenics (Fig.
6). Since overproduction of either PEPC or ME alone did
not lead to appreciable stunting and overproduction of ME
in addition to PEPC did not affect photosynthesis (Fig.
7B), it is possible that PEPC and ME together catalyse
a futile pathway which wastes metabolic energy.
As described in the Results, PPDK and MDH undergo
strict activity regulation by light, and they are completely
inactivated in darkness in transgenic rice leaves, while
PEPC is active throughout the day. The C3-specific ME
may also be active both in the light and in darkness, as
judged by its enzyme characteristics. The C4-specific
isoform has a pH optimum of around 8 and it is activated
by light through the elevation of pH and the increase in an
essential cofactor Mg2+ in the stroma (Edwards and
Andreo, 1992; Drincovich et al., 2001). In addition, its
sensitivity to inhibition by the substrate malate depends
on pH: it is inhibited by malate at pH 7 while it becomes
insensitive at pH 8, thus exhibiting a high activity only in
the light in C4 plants (Edwards and Andreo, 1992).
Activity regulation through a change in pH is possible for
the C3-specific isoform, for which a pH optimum of 7.8
has been reported for the recombinant maize protein
(Saigo et al., 2004). Unlike the C4-specific ME, however,
the C3-specific isoform is not inhibited by malate at pH 7
(Drincovich et al., 2001; Edwards and Andreo, 1992),
remaining active even in darkness. The level of NADP+ in
the stroma in darkness would be high enough to support
the decarboxylation reaction of overproduced ME, and
1808 Taniguchi et al.
moreover, direct uptake of malate is feasible. When PEPC
is overproduced together with ME, it raises the level of
malate inside the cell so that overproduced ME can
support appreciable flux.
The maize PEPC in transgenic rice leaves is phosphorylated and in the more active form during a period from 2 h
before the beginning of the dark period until 2 h after
the onset of the light period (Fukayama et al., 2003).
Therefore, the putative wasteful pathway supported by
PEPC and ME seems to be active at night and also for
some time after sunrise and before sunset. The stunting
caused by overproduction of PEPC with ME would thus
be inevitable unless the phosphorylation pattern of PEPC
in rice leaves were to be modified. By contrast, stunting
seems to be much less marked in other transgenic C3
plants such as barley, cucumber, Arabidopsis, and Lotus
japonicus, in which PEPC is phosphorylated in the
daytime (H Fukayama et al., unpublished results). Previous studies on transgenic potato and tobacco plants
overproducing PEPC together with ME (Lipka et al.,
1999; Häusler et al., 2001) did not report stunting of these
transformants. Even in rice plants, the stunting might be
greatly mitigated when the C3-specific PEPC is used as
the transgene source, as this protein is more sensitive to
inhibition by malate than the C4-specific isoform (Svensson et al., 2003). In any case, the activity regulation of
PEPC is essential for the growth of transgenic C3 plants.
As demonstrated previously, overproduction of PEPC
almost void of activity regulation leads to serious stunting
of transgenic Arabidopsis and potato plants (Rademacher
et al., 2002; Chen et al., 2004; for a review see Miyao and
Fukayama, 2003).
Taken together, we consider that rice is not good plant
material for an approach to introduce the C4-like pathway
with respect to increased production. Even so, this study
does not eliminate the possibility of improving photosynthesis of C3 plants by this approach. Four homozygous
lines of the quadruple transformants used in this study
unexpectedly had almost the same activities of MDH and
ME (Table 1). It remains to be determined whether
photosynthesis could be substantially improved by changing the relative level of expression of the introduced
enzymes, as well as the nature of a metabolic pathway(s)
operating in the quadruple transformants.
Acknowledgements
The authors are grateful to Professors Myroslawa Miginiac-Maslow,
CNRS/University Paris-Sud, France, Katsura Izui, Kinki University,
Japan, and Toru Hisabori, Tokyo Institute of Technology, Japan, for
their generous gifts of antibodies, the cDNA clone and the
recombinant protein. They are also thankful to Emeritus Professor
Kuni Ishihara, Tokyo University of Agriculture and Technology,
Japan, and to Professors Amane Makino, Tohoku University, Japan,
and Hideaki Usuda, Teikyo University, Japan, for their helpful
discussion and encouragement throughout the study and to Drs Julie
and Robert T Furbank, CSIRO, Australia, for their careful reading
of the manuscript. This work was supported by a grant from the
Ministry of Agriculture, Forestry and Fisheries of Japan (Green
Technology Project IP-3001) to MM.
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