Download Overexpression of C4-cycle enzymes in transgenic C3 plants: a

Journal of Experimental Botany, Vol. 53, No. 369, pp. 591–607, April 2002
REVIEW ARTICLE
Overexpression of C4-cycle enzymes in transgenic
C3 plants: a biotechnological approach to improve
C3-photosynthesis
Rainer E. Häusler1,3, Heinz-Josef Hirsch2, Fritz Kreuzaler2 and Christoph Peterhänsel2
1
2
Botanik II, Botanisches Institut der Universität zu Köln, Gyrhofstrasse 15, D-50931 Cologne, Germany
Institut für Biologie I, RWTH, Worringer Weg 1, D-52074 Aachen, Germany
Received 8 November 2001; Accepted 18 December 2001
Abstract
The process of photorespiration diminishes the
efficiency of CO2 assimilation and yield of C3-crops
such as wheat, rice, soybean or potato, which are
important for feeding the growing world population.
Photorespiration starts with the competitive inhibition of CO2 fixation by O2 at the active site of ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco)
and can result in a loss of up to 50% of the CO2 fixed
in ambient air. By contrast, C4 plants, such as maize,
sugar cane and Sorghum, possess a CO2 concentrating mechanism, by which atmospheric CO2 is bound
to C4-carbon compounds and shuttled from the
mesophyll cells where the prefixation of bicarbonate
occurs via phosphoenolpyruvate carboxylase (PEPC)
into the gas-tight bundle-sheath cells, where the
bound carbon is released again as CO2 and enters
the Calvin cycle. However, the anatomical division
into mesophyll and bundle-sheaths cells (‘Kranz’anatomy) appears not to be a prerequisite for the
operation of a CO2 concentrating mechanism. Submerged aquatic macrophytes, for instance, can induce
a C4-like CO2 concentrating mechanism in only one
cell type when CO2 becomes limiting. A single cell
C4-mechanism has also been reported recently for a
terrestrial chenopod. For over 10 years researchers
in laboratories around the world have attempted to
improve photosynthesis and crop yield by introducing
a single cell C4-cycle in C3 plants by a transgenic
3
approach. In the meantime, there has been substantial progress in overexpressing the key enzymes
of the C4 cycle in rice, potato, and tobacco. In this
review there will be a focus on biochemical and
physiological consequences of the overexpression
of C4-cycle genes in C3 plants. Bearing in mind that
C4-cycle enzymes are also present in C3 plants, the
pitfalls encountered when C3 metabolism is perturbed by the overexpression of individual C4 genes
will also be discussed.
Key words: Biotechnology, crop yield, transgenic C3 plants.
Introduction
Photorespiration decreases the efficiency of
CO2 assimilation in C3 plants
Most of our crops, such as wheat, rice, soybean or
potato are classified as C3 plants as the first product of
atmospheric CO2 fixation is the 3-carbon compound
3-phosphoglycerate (3-PGA), which is produced in the
Calvin cycle by Rubisco (the only enzyme capable of net
carbon assimilation) in the chloroplast stroma. However,
competition of O2 with CO2 at the active site of Rubisco
(Chen and Spreitzer, 1992; Jordan and Ogren, 1984)
results in a loss of up to 50% of the carbon fixed in
a process known as photorespiration (Ogren, 1984).
To whom correspondence should be addressed. Fax: q49 221 470 5039. E-mail: [email protected]
Abbreviations: C*, CO2 compensation point in the absence of dark respiration in the light; CA, carbonic anhydrase; NAD(P)-MDH, NAD(P)-dependent
malate dehydrogenase; NAD(P)-ME, NAD(P)-dependent malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate
carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PEPS, phosphoenolpyruvate synthetase; 3-PGA, 3-phosphoglycerate; 2-PG,
2-phosphoglycollate; PFD, photon flux density; PPDK, pyruvate, orthophosphate dikinase; PPT, phosphoenolpyruvate/phosphate translocator; Rd,
dark respiration in the light; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; TPT, triose phosphate/phosphate translocator.
ß Society for Experimental Biology 2002
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Oxygenation of ribulose-1,5-bisphosphate (RubP) severely
diminishes the efficiency of CO2 assimilation in C3 plants
in ambient air and results in the formation of 3-PGA as
well as 2-phosphoglycollate (2-PG). The latter is metabolized in three compartments of the leaf cell, the chloroplast, the peroxysomes and the mitochondria, involving
numerous enzymatic reactions and transport processes
(Fig. 1). The overall photorespiratory cycle is also linked
to amino acid metabolism in that glycine, serine, glutamate, and glutamine are metabolized at high rates (Keys
et al., 1978). Both CO2 and ammonia are released at equal
rates in the reaction catalysed by the mitochondrial
glycine decarboxylase complex (Oliver, 1994). The loss
of CO2 during photorespiration is reflected in a CO2
compensation point (C) of CO2 assimilation of between
40–60 ml l 1 CO2 in the intercellular air space. At the
CO2 compensation point, net CO2 assimilation equals
CO2 release through photorespiration and mitochondrial
respiration in the light. In high CO2 anduor low O2 the
oxygenase activity of Rubisco is virtually absent, the flux
through the photorespiratory carbon cycle negligible and
the CO2 compensation point close to zero.
There are numerous reports on the improvement of
growth and crop yield of C3 plants in an atmosphere
containing elevated CO2 (Arp et al., 1998; Besford, 1990;
Chen et al., 1997; Teramura et al., 1990). This is
mainly based on a faster biomass production due to an
increase in CO2 assimilation rates and a suppression of
photorespiration.
C4 plants have developed strategies to concentrate
CO2 in the vicinity of Rubisco
During the evolution of higher plants, adaptations
to low water supply anduor hot environments have
developed independently several times (Edwards et al.,
2001; Kellogg, 1999; Ku et al., 1996). Plants, which display
C4-metabolism, release CO2 at high rates in the vicinity
of Rubisco and thereby increase the ratio of RubP
carboxylationuoxygenation substantially (Leegood, 1997,
2002). This strategy prevents major losses of CO2 by
photorespiration and is accompanied by an increase in
the water and nitrogen use efficiency compared to C3
plants (Sage and Pearcy, 1987). Common C4 plants are
characterized by the so-called ‘Kranz’-anatomy with
mesophyll cells surrounded by relatively thin cell walls
and bundle-sheath cells surrounded by thick cell walls.
This anatomical separation into different cell types is
Fig. 1. The photorespiratory carbon and nitrogen cycle typical for C3 plants in ambient air. Photorespiration starts with the oxygenase reaction of
Rubisco (1). (2) Phosphoglycollate phosphatase, (3) glycollate oxidase, (4) glutamate : glyoxylate aminotransferase, (5) serine : glyoxylate
aminotransferase, (6) glycine decarboxylase, (7a, b) NAD malate dehydrogenase, (8) hydroxypyruvate reductase, (9) glycerate kinase, (10) glutamine
synthetase, (11) glutamate synthase.
Biotechnology and C3 photosynthesis
accompanied by a spatial separation of the prefixation
of atmospheric CO2 in the mesophyll cells followed by the
release of CO2 and its refixation via the C3 (Calvin) cycle
in the bundle-sheath cells. Moreover, in order to keep
intercellular diffusion ways short, mesophyll cells are in
close proximity to the bundle-sheath (Dengler and
Nelson, 1999). The first step in the C4-cycle is the
carboxylation of PEP by phosphoenolpyruvate carboxylase (PEPC) in the cytosol of the mesophyll cells using
HCO3 as the inorganic carbon substrate (Cooper and
Wood, 1971; O’Leary, 1982). This yields the C4 dicarboxylic acid oxaloacetate (OAA), which is either reduced
to malate or transaminated to aspartate and is then
transported into the bundle-sheath cells, where CO2 is
released at high rates by decarboxylating enzymes.
Depending on the type of C4 plants, CO2 is released
either by chloroplastic NADP malic enzyme (ME),
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mitochondrial NAD-ME, or cytosolic phosphoenolpyruvate carboxykinase (PEPCK) (Hatch and Osmond,
1976; Leegood, 2002) (Fig. 2). Since bundle-sheath cells
have low gas permeability, the CO2 concentration in
solution is drastically increased, which causes a suppression of the Rubisco oxygenase activity and, consequently,
photorespiration (Leegood, 1997). Pyruvate released by
NADP-ME or NAD-ME is transferred back into the
mesophyll chloroplasts where the inorganic carbon
acceptor PEP is regenerated by pyruvate, Pi dikinase
(PPDK). The reaction, catalysed by PPDK, consumes in
addition two ATP per CO2 assimilated. One ATP is
directly required by PPDK, the second ATP is needed
for the conversion of the reaction product AMP into
ADP catalysed by adenylate kinase (Fig. 2). The C4
strategy permits high rates of CO2 assimilation at a
relatively small stomatal aperture and thereby increases
Fig. 2. CO2 concentrating mechanism in a NADP malic enzyme C4 plant, such as maize, sugar cane or Sorghum. CO2 is converted to HCO3
by carbonic anhydrase (1) in the cytosol of the mesophyll cells and fixed by oxygen-insensitive PEPC (2). The oxaloacetate formed is imported into
the stroma of the mesophyll chloroplasts and reduced by NADP-MDH (3) using redox equivalents from non-cyclic electron transport. Malate is
exported from the stroma in counter exchange with OAA catalysed by a malateuOAA transporter (4). In the mesophyll cell the concentration of
malate is high, which allows diffusion along a concentration gradient to the bundle-sheath cells. Malate enters the bundle-sheath chloroplasts and
is subjected to oxidative decarboxylation by NADP-ME (5). As bundle-sheath cells are gas tight, the high rate of oxidative malate decarboxylation
results in a steep increase in the CO2 concentration in the vicinity of Rubisco (6) and hence a suppression of the oxygenase activity and
photorespiration. As bundle-sheath chloroplasts of NADP-ME C4 plants lack photosystem II and hence the capacity for non-cyclic electron transport,
NADPH formed by NADP-ME is utilized for the reduction of 50% of 3-PGA formed by Rubisco. The residual 3-PGA is exported by a C4-type
TPT (7a) and is transferred to the mesophyll chloroplasts, imported into the stroma via the TPT (7b) and reduced to triose phosphates. Pyruvate, the
product of malate decarboxylation also diffuses into mesophyll cells, enters the chloroplasts via a pyruvate transporter (8) and the primary inorganic
carbon acceptor PEP is regenerated by PPDK (9). The PPi released by this reaction is cleaved by pyrophosphatase (10) and the AMP converted to
ADP by adenylate kinase (11). Hence, for the regeneration of PEP an additional two ATP are required. PEP is exported from the chloroplast via the
PPT (12). For the sake of clarity not all cofactors are shown.
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the water use efficiency. Moreover, due to an almost
complete suppression of RubP oxygenation the CO2
compensation point is close to zero or very low as
compared to C3 plants.
C4 plants lacking ‘Kranz’ anatomy
Since the discovery of the C4 cycle some 36 years ago the
spatial separation into mesophyll cells and bundle sheath
cells was thought to be a prerequisite for an efficient CO2concentrating mechanism. It is therefore quite amazing
that the submerged aquatic plant Hydrilla verticillata was
identified as being capable of inducing a C4-like metabolism (Fig. 3A), but lacking the ‘Kranz’-anatomy typical
for C4 plants (Holaday and Bowes, 1980; Magnin et al.,
1997; Salvucci and Bowes, 1981, 1983; Spencer et al.,
1996). The switch from C3- to C4-like metabolism in
H. verticillata is triggered by low CO2 concentrations
(i.e. at high water temperatures) resulting in an increase
in PEPC, ME and PPDK activities, which causes a substantial drop in the CO2 compensation point from around
40 ml l 1 in the C3-state down to below 10 ml l 1 in the
C4-induced state (Spencer et al., 1996; Reiskind et al.,
1997) (Fig. 3A). Further evidence for a C4-like metabolism without ‘Kranz’ anatomy emerged for other
aquatic species such as Egeria densa (Casati et al., 2000)
and (probably) Elodea canadensis (de Groote and
Kennedy, 1977). Moreover, apart from the inducible
C4-photosynthesis these aquatic species exhibit a
pH-polarity of their leaf surfaces. The steady-state pH
of the adaxial surface is acidic (pH 4.0), whereas the
abaxial side of the leaf is alkaline (pH 10.0) (van Ginkel
et al., 2001). An acidic environment allows higher CO2
concentrations at equilibrium despite a drop in the total
inorganic carbon in the surrounding water. This supplementary mechanism to increase the availability of
external CO2 is also apparent in the C4-induced state of
H. verticillata.
Fig. 3. (A) Induced C4 cycle in the submerged aquatic plant Hydrilla verticillata without ‘Kranz’ anatomy. (1) CA, (2) PEPC, (3) NAD-MDH,
(4) NADP-MDH, (5) NADP-ME, (6) Rubisco, (7) PPDK, (8) PPT. (B) Genetically engineered single cell CO2 concentrating mechanism in transgenic tobacco plants. The transformants contain all combinations of C4-cycle enzymes (PEPC, PEPCK, NADP-ME, PPDK or PEPS as well as the
PPT). For the sake of clarity not all cofactors are shown.
Biotechnology and C3 photosynthesis
Interestingly, single cell C4 photosynthesis appears not
to be restricted to aquatic macrophytes. Recent determinations of d13C values (a measure for the activity of
PEPC compared to Rubisco, which discriminates 13CO2
in favour of 12CO2) combined with anatomical studies
revealed that the terrestrial chenopod Borszczowia aralocaspica (a halophyte with succulent leaves adapted to a
semi-dry environment) is likely to carry out C4 metabolism in only one cell type (Freitag and Stichler, 2000).
Moreover, the individual chlorenchyma cells of B. aralocaspica carry two types of chloroplasts with a distinct
enzyme equipment and capability to produce starch.
Immunolocalization of enzyme proteins revealed that
Rubisco (as well as starch) is localized in chloroplasts of
the basal part of the chlorenchyma cells (i.e. closer to
the vascular bundles), whereas PPDK is localized in the
chloroplasts of the distal parts of the cells (i.e. closer to
the intercellular air space). Like Rubisco, NAD malic
enzyme, which appears to act as the decarboxylating
enzyme is also more abundant in the mitochondria of
the basal parts of the chlorenchyma cells. PEPC is equally
distributed throughout the cytosol (Vozesenskaya et al.,
2001).
Crassulacean acid metabolism (CAM) plants also exhibit
a single cell CO2-concentrating mechanism
A different strategy of a single cell CO2-concentrating
mechanism is realized in Crassulacean acid metabolism
(CAM) plants, which also show an adaptation to a
minimized water supply. In CAM plants both the
prefixation of HCO3 by PEPC and the release of CO2
by decarboxylating enzymes is temporally separated
(Osmond, 1978; Cockburn et al., 1979; Spalding et al.,
1979). HCO3 is prefixed in the dark by PEPC at low
temperatures and open stomata and malic acid, which
accumulates in the vacuole during the night is decarboxylated during the next day, when stomata are closed, either
by cytosolic NADP-ME in concert with mitochondrial
NAD-ME or by cytosolic PEPCK. This mechanism
efficiently reduces the loss of water and also results in a
high CO2 environment in the mesophyll during the day
and thereby suppresses photorespiration.
Attempts to introduce a single cell CO2-concentrating
mechanism into terrestrial crops
In laboratories around the world attempts to introduce
single cell C4-like features into terrestrial C3 plants by
a transgenic approach are in progress (Matsuoka et al.,
2001). It is believed that the introduction of an intracellular CO2 pump might improve the efficiency of C3
photosynthesis by a substantial suppression of photorespiration. There has been progress in single, double and
multiple overexpressions of C4-cycle enzymes in C3 crops.
In particular, very high expression levels could be achieved
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in transgenic rice (Ku et al., 1999). However, physiological alterations caused by this approach have been
addressed only in a few reports. As C4-cycle enzymes
are also common in C3 plants, although with much
smaller activities, the introduction of individual C4-cycle
genes might perturb not only primary metabolism, but
in certain cases also secondary metabolism (Häusler
et al., 2001).
Although a single cell C4-like system appears to
operate in aquatic macrophytes and also in a terrestrial
chenopod adapted to semi-dry climates, it is still an open
question whether a biotechnologically inserted CO2
pump in terrestrial C3 crops can efficiently increase the
carboxylationuoxygenation ratio of Rubisco. It is questionable whether the additional CO2 released from the
products of PEP carboxylation can be retained within the
mesophyll or whether it is just lost to the atmosphere,
because of high diffusion rates of CO2 in the airspace of
the leaves (Leegood, 2002). In order to match the requirements of the metabolic environment of individual C3 crop
species, future research might also focus more on genetically engineered C4-cycle enzymes driven by more specific
promoters. In this review there will be a focus on biochemical and physiological consequences of the overexpression of C4-cycle genes in the C3 plants Solanum
tuberosum and Nicotiana tabacum. The progress in the
genetic manipulation of rice plants, particularly the
achievement of extremely high expression levels will
be dealt with in a second review.
The role of C4-cycle enzymes in C3 plants
Exceptionally, all enzymes and metabolite transporters
involved in the C4 pathway also occur in C3 plants,
although at much lower activities and different tissue
specificities. Before the question as to the feasibility of the
approach to establish a C4-like cycle in C3 plants is
tackled, one has to be aware of the physiological
functions of the C3 forms of the individual enzymes and
transporters summarized in Table 1.
PEPC [ EC 4.1.1.31]
As in C4 plants, PEPC in C3 plants is subject to complex
regulation by metabolites and covalent modification by
reversible phosphorylation (Andreo et al., 1987; Van Quy
et al., 1991; Duff and Chollet, 1995; Zhang et al., 1995;
Chollet et al., 1996). In certain types of heterotrophic
tissues from developing fruits and seeds of C3 plants, the
CO2 respired is recaptured by PEPC. These tissues contain relatively high PEPC activities (reviewed by Latzko
and Kelly, 1983). In leaves of C3 plants one role of
PEPC is the supply of carbon skeletons for amino acid
biosynthesis following nitrate assimilation (Andrews,
1986; Melzer and O’Leary, 1987) (Fig. 4). During nitrate
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Table 1. Some proposed functions of‘C4-cycle enzymes’ and transporters in C3 plants
Enzymes
PEPC
NADP-ME
Location
Proposed metabolic function
Non-green tissues
Leaves
Recapture of CO2 respired.
Anaplerotic supply of carbon skeletons for amino acid biosynthesis.
Buffering cytosolic OH formation during nitrate reduction by malic acid formation.
Formation of malic acid during stomatal opening.
De-acidification of vacuoles, provision of reducing equivalents and carbon skeletons for gluconeogenesis.
In combination with PEPC involved in pH-stat. In combination with NAD-MDH involved in
NADHuNADPH conversion.
Provision of reducing equivalents and carbon skeletons for fatty acid biosynthesis.
In vascular bundles, provision of reducing equivalents for lignin biosynthesis.
Stress responses.
Together with PEPC involved in anaplerotic provision of carbon skeletons for amino acid biosynthesis.
Reduction of OAA in the chloroplast. Shuttling excessive redox equivalents (malate valve)
into the cytosol (mitochondria).
Gluconeogenetic PEP production from OAA.
Involvement in secondary metabolism.
Largely unresolved. (PEP production for the shikimate pathway?)
Gluconeogenetic PEP generation from pyruvate during stomatal closure.
Stomatal guard cells
Fruits
Seeds
Leaves
NAD-ME
NADP-MDH
Leaves
Leaves
PEPCK
Non-green tissues
Trichomes
PPDK
Stomatal guard cells
Transporters
Pyruvate
MalateuOAA
PPT
Leaves
Fatty acid and branched chain amino acid biosynthesis.
Malate valve, see NADP-MDH.
Provision of PEP for the shikimate pathway inside the chloroplast (also fatty acid biosynthesis).
Fig. 4. Role of PEPC in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3 plants. (1) Triose
phosphateuphosphate translocator, (2) PEPC, (3) NAD-MDH, (4) NADP-MDH, (5) pyruvate kinase, (6) NAD-ME, (7) pyruvate dehydrogenase,
(8) NAD isocitrate dehydrogenase, (9) NADP isocitrate dehydrogenase. For the sake of clarity not all cofactors are shown.
Biotechnology and C3 photosynthesis
reduction in the cytosol OH ions are generated. As
both processes, nitrate reduction and malate formation,
are closely correlated the production of OAA via PEPC
and the consecutive formation of malic acid may buffer
cytosolic pH (Martinoia and Rentsch, 1994). Moreover,
PEPC also plays an important role in stomatal movement. An increased influx of potassium ions into the
guard cells during stomatal opening and the concomitant
efflux of protons via the plasma membrane-bound
ATP-dependent proton pump would result in a rapid
alkalization of the cytosol (Raschke et al., 1988). This
is counteracted by the synthesis of malic acid from neutral
sugars or transitory starch via PEPC. For instance, a
substantial decrease in the stomatal aperture has been
reported for Vicia faba after the application of DCDP
(3,3-dichloro-2-dihydroxyphosphinoyl-2-propeoate) a
specific inhibitor of PEPC (Asai et al., 2000).
NADP-ME [ EC 1.1.1.40]
NADP-ME contributes to a huge variety of metabolic
pathways in green and non-green tissues of C3 plants as
reviewed previously (Edwards and Andreo, 1992). It is
found in leaves, etiolated tissues, seeds, roots, fruits, and
tubers (potato) in the chloroplast as well as in the cytosol.
In fruits, NADP-ME is involved in ripening and in the
de-acidification of the vacuole as well as in the provision
of reducing equivalents and carbon skeletons for sucrose
biosynthesis via gluconeogenesis. This is comparable to
the situation in CAM plants in the light. Together with
PEPC, NADP-ME might also serve as a pH-stat and in
combination with NAD malate dehydrogenase (MDH)
could be involved in the conversion of NADH to NADPH.
In oil-storing tissues, such as seeds of rapeseed or wheat
germ, NADP-ME is engaged in the proliferation of
reducing power and carbon skeletons for fatty acid
biosynthesis.
Leaves of solanaceous species, such as potato and
tobacco, contain substantial activities of cytosolic
NADP-ME (Knee et al.,1996). There are indications that
the cytosolic enzyme is associated with vascular bundles,
particularly with developing xylem and internal phloem
(Schaaf et al., 1995) suggesting that NADPH produced by
this reaction is required for lignin biosynthesis. Moreover,
NADP-ME appears to be involved in stress responses
(Casati et al., 1999). In leaf tissues, the expression
of NADP-ME is increased severely by wounding
in combination with glutathionine treatment (Schaaf
et al., 1995).
NAD-ME [ EC 1.1.1.39]
NAD-ME is localized in the mitochondria and in C3
plants is involved in the anaplerotic carbon supply for
amino acid biosynthesis (Fig. 4). Malate formed by PEPC
can either be used in the citric acid cycle or it can be
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decarboxylated to provide pyruvate and subsequently
acetyl-CoA (Douce and Neuburger, 1989).
NADP-MDH [ EC 1.1.1.82]
In leaves of C3 plants, NADP-MDH plays a central role
in shuttling excessive redox equivalents from the chloroplast into the cytosol (Scheibe, 1987). This so-called
malate valve operates at high light intensities, when
electron transport rates and NADPH generation exceed
the demands for CO2 assimilation and thereby prevents
overreduction of the stroma anduor increased Mehlerperoxidase reaction (Polle, 1996). NADP-MDH is
subjected to reversible activation by thioredoxin-m and
thereby responds to the redox state of the stroma.
PEPCK [ EC 4.1.1.39]
As reviewed recently (Leegood et al., 1999) plant PEPCK
is a cytosolic enzyme and is basically involved in ATPdependent gluconeogenetic PEP production from OAA.
In C3 plants, high PEPCK activities were detected during
germination of oil-storing seeds, during fruit ripening, in
phloem-associated cells, and in developing seeds. PEPCK
is also found in trichomes of tobacco and cucumber
leaves and is believed to be involved in the production
of secondary metabolites. There also appears to be a
function of PEPCK in plant defence reactions.
PPDK [ EC 2.7.9.1]
In C3 plants, low PPDK activities were reported for
a number of different tissue types and, depending on
the species, it is found only in chloroplasts or in both
chloroplasts and the cytosol (Aoyagi and Bassham, 1984;
Nomura et al., 2000). The function of PPDK in C3 plants
is less clear. However, for guard cells of Vicia faba it has
been proposed that chloroplastic PPDK in concert with
cytosolic NADP-ME could play a role in gluconeogenetic
PEP generation from pyruvate during stomatal closure
(Schnabl, 1981).
Metabolite transporters
High fluxes of C4 photosynthesis are accompanied by
transport processes across membranes, particularly the
inner chloroplast envelope. In the mesophyll cells,
pyruvate enters the chloroplast via a pyruvate Hq
symporter as a substrate for PPDK (Flügge et al.,
1985). Likewise, PEP generated within the chloroplasts
has to get access to the cytosol via a PEPuphosphate
translocator (PPT). Furthermore, there are significantly
high rates of OAAumalate exchange required for a fast
production of malate inside the mesophyll chloroplasts.
Moreover, in NADP-ME C4 plants, malate must
enter the bundle-sheath chloroplasts. Following oxidative decarboxylation of malate by NADP-ME, pyruvate
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formed by this reaction has to be exported from the
chloroplast.
Pyruvate imported into chloroplasts of C3 plants
can be used for fatty acid biosynthesis or the production
of branched chain amino acids. A rapid malateuOAA
exchange across the inner envelope is required for the
malate valve in C3 plants. However, the PPT is the only
metabolite transporter also involved in C4 metabolism,
which has been cloned from a variety of non-C4-tissues
and is found at low activities in the inner envelope
membrane of chloroplasts and non-green plastids of
C3 plants as well as in maize roots (Fischer et al., 1997).
Most likely, the PPT provides plastids with PEP as a substrate for the shikimate pathway (Streatfield et al., 1999).
Import of PEP (rather than export) is a prerequisite for
aromatic amino acid biosynthesis, which is localized
entirely within this organelle and results in a huge variety
of secondary products downstream of phenylalanine
(Schmid and Amrhein, 1995).
The need for an accelerated metabolite exchange
across the chloroplast envelope as observed in C4 plants
ought to be considered as well in attempts to introduce
C4-like features into C3 plants. Therefore genes encoding
the relevant plastidial metabolite transporters, apart from
the PPT, should be identified and isolated.
Progress to date in the physiological and
biochemical characterization of C3 plants
overexpressing C4-cycle genes
As C4-cycle enzymes (and metabolite transporters) have
distinct functions in C3 plants (Table 1), an increase in
their activities by individual overexpression is likely to
perturb metabolism or to trigger compensational changes
in metabolic fluxes. Compensational changes in metabolism as a response to the overexpression of C4-cycle
enzymes are interesting to study, as these changes may
point to not yet unravelled regulatory interrelationships
between different metabolic pathways. It is conceivable
that a fully operational single cell C4-cycle circumvents
such metabolic perturbations.
Overexpression of PEPC is the first step for establishing
a C4 cycle in C3 plants
The efficient fixation of atmospheric CO2 by PEPC is
a prerequisite for the insertion of a single cell C4 cycle in
C3 plants. The first cDNAs of the C4-type PEPC
sequences for cloning were obtained from maize (Izui
et al., 1986) and Flaveria trinervia (Poetsch et al., 1991). A
successful introduction of the maize PEPC into transgenic
tobacco plants under the control of its own promoter or
the mesophyll-specific promoter of the chlorophyll aub
binding protein gene (cab) was achieved (Hudspeth et al.,
1992) and later on by using the constitutively expressing
cauliflower mosaic virus (CaMV) 35S promoter (Kogami
et al., 1994; Benfey and Chua, 1990). In these types of
transgenic plants PEPC activity was increased slightly
more than 2-fold. Apart from an increase in the malate
contents, there were no apparent effects on the rate of
CO2 assimilation. However, in contrast to the wild type,
the quantum yield for CO2 assimilation appeared to be
unaffected by increasing temperatures in one transgenic
line (Kogami et al., 1994). The lack of decrease in the
quantum yield with increasing temperature suggested a
refixation of respired CO2 by PEPC in the transgenics.
However, the CO2 compensation point measured in a
sealed chamber remained unaffected in transgenic
tobacco plants compared to the control plants
(Hudspeth et al., 1992; Kogami et al., 1994).
In rice, high expression of the maize PEPC was
achieved recently using the complete maize PEPC gene
including exons and introns and its own promoter for
transformation and resulted in a 110-fold increase in
PEPC activity measured in vitro (Ku et al., 1999). The
reported decline in O2 inhibition of CO2 assimilation
would have been consistent with an attenuation of photorespiration. However, a more detailed analysis of these
plants suggested that this effect was likely to be based on
phosphate limitation of photosynthesis under conditions
that promote high photosynthetic fluxes (Fukayama et al.,
2001; Matsuoka et al., 2000). Still, it is hard to conceive
as to why phosphate limitation of photosynthesis should
occur with an increase in PEPC activitiy. Provided that
PEP is generated via glycolysis starting from triose
phosphates exported from the chloroplast in the light,
the phosphate stoichiometry would be balanced as for
each triose phosphate exported one phosphate is released
from PEP by PEPC and could serve as a counter exchange
substrate regardless of whether the flux into sucrose biosynthesis was slowed down or not (Fig. 4). If phosphate
limitation was the cause for lower CO2 assimilation rates
under optimum conditions, it is therefore more likely
that it occurs at the site of cytoplasmic glyceraldehyde3-phosphate dehydrogenase, leading to the subsequent
formation of ATP by phosphoglycerate kinase. This
reaction sequence could (at least temporarily) deplete
cytoplasmic phosphate pools required for triose phosphate counter exchange. It is also conceivable that
additional OAA formed by PEPC is reduced to malate
in the stroma and hence competes with reducing equivalents needed for 3-PGA reduction. However, to the
knowledge of the authors, neither of these alternative
explanations has been addressed experimentally.
As potato was chosen as a model plant for one laboratory study, cDNAs and genomic clones of the endogenous C3-type PEPC were isolated (Merkelbach et al.,
1993). However, it was then decided to use PEPC from
a bacterial source rather than the endogenous enzyme
from potato or other plants. Bacterial enzymes lack, for
Biotechnology and C3 photosynthesis
instance, the regulatory properties of plant enzymes, such
as the covalent modification by phosphorylation. Overexpression of two bacterial PEPC genes from Escherichia
coli and Corynebacterium glutamicum under the control of
the CaMV 35S promoter resulted eventually in a 5-fold
increase in PEPC activity in potato leaves with the enzyme
from C. glutamicum (Gehlen et al., 1996). Depending on
the composition of the in vitro assay an increase of up to
20-fold could be determined (Häusler et al., 2001). Similar
to the report on the PEPC overexpressors of tobacco
(Hudspeth et al., 1992; Kogami et al., 1994), the absolute
rates of photosynthetic CO2 assimilation and electron
transport were not severely affected in potato plants
overexpressing the bacterial PEPC or in plants with an
antisense repression of the endogenous enzyme. Likewise,
the CO2 compensation point (C), determined in a closed
chamber was unaffected in the trangenics. However, dark
CO2 release after illumination was considerably increased
in PEPC overexpressors and diminished in the antisense
plants (Gehlen et al., 1996). This suggested that PEPC
affects the rate of respiratory CO2 release. More recently
it could be demonstrated that the CO2 compensation
point (C*) independent of dark respiration in the light
(Rd) (determined according to Brooks and Farquhar,
1985), was appreciably diminished in PEPC overexpressors and slightly increased in potato plants with an antisense repression of the endogenous PEPCs (Fig. 5)
(Häusler et al., 1999). Rd (the rate of dark respiration
in the light) was increased in a range of PEPC overexpressors and slightly decreased in the antisense plants.
As C* solely reflects the kinetic properties of Rubisco
(Brooks and Farquhar, 1985), but not accounts for
carboxylation of PEP by O2-insensitive PEPC, the changes
in C* were interpreted as changes in the CO2uO2 ratio in
the vicinity of Rubisco, which was most likely due to
an increased release of CO2 from products of PEP carboxylation. In theory, even more CO2 can be deliberated
from PEP carboxylation products than is actually fixed
via PEPC in the form of HCO3 . If, for instance, malate
were completely oxidized in the citric acid cycle (Fig. 4),
this would result in the release of four CO2 for each
carboxylation of PEP on the expense of photoassimilates.
However, all intermediate stoichiometry of HCO3 uptake
and CO2 release might occur as well. An enhanced CO2
release by decarboxylating enzymes would result in an
efficient HCO3 uCO2 pump (provided that the activity
of cytosolic carbonic anhydrase is absent or low), which
could increase the CO2 concentration in the mesophyll
cells as well as in the vicinity of Rubisco leading to a
slight inhibition of photorespiration. These theoretical
considerations were reinforced by the observation that
overexpression of PEPC induced the endogenous cytosolic NADP-ME of potato plants by a factor of 4–6
(Häusler et al., 2001). This induction was accompanied by
increased activities of a putative cytosolic pyruvate kinase
599
Fig. 5. Typical dependencies of CO2 assimilation from the intercellular
CO2 concentration (Ci ) required for the determination of C* and Rd
in control potato (A), a transformed line overexpressing PEPC from
C. glutamicum (B) and a transformed line with antisense repression of
the endogenous PEPC (C). Gas exchange measurements were done at
22 8C and a relative humidity of 30% at limited PFDs (11, 74, 100 mmol
m 2 s 1) and a saturated PFD (320 mmol m 2 s 1). The intercept of the
AuCi dependencies indicate C* (on the Ci-axis) and Rd (on the A-axis).
The error for the calculation of the intercept for the individual sets
of measurements was below 5%. (This figure is taken from Häusler
et al., 1999.)
and a presumably cytosolic NADP isocitrate dehydrogenase (Chen, 1998) suggesting that an increased activity
of cytosolic NADP-ME in potato leaves initiates
CO2 release from PEP carboxylation products (Häusler
et al., 2001).
In another approach to increase PEPC activity, the
endogenous potato enzyme was modified in a way that
the phosphorylation site was mutated, which yielded an
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Häusler et al.
enzyme with a higher affinity for PEP and lowered
sensitivity towards malate inhibition. A detailed report
on the analysis of these plants is in preparation by
Rademacher et al. Transgenic potato plants with a
4 –5-fold increase in the activity of the modified version
of potato PEPC also exhibited a pleiotropic increase
in endogenous cytosolic NADP-ME (Häusler et al.,
2001).
The sole increase in PEPC activity therefore
appears to perturb metabolic fluxes and can lead to
a waste of photoassimilates (T Rademacher, personal
communication).
Stomatal movement is affected in PEPC transgenics
of potato
PEPC from C. glutamicum was expressed under the
control of the CaMV 35S promoter. Hence the enzyme
is constitutively expressed in most tissues and it was
very likely to be also present in stomatal guard cells.
Interestingly, stomatal opening was accelerated in PEPC
overexpressors and delayed in PEPC antisense plants
compared to the wild type (Gehlen et al., 1996). The average half time for stomatal opening was 3 min, 9 min, and
6 min for PEPC overexpressors, the antisense plants and
the wild type, respectively. Furthermore, oscillations in
stomatal conductance were also observed in PEPC
overexpressors. These side-effects, underlining the role
of PEPC during stomatal movement (see also Asai et al.,
2000), deserve a more thorough investigation.
Pleiotropic effects on secondary metabolism
Overexpression of PEPC from bacterial and plant sources
not only affects primary metabolism, but also appears
to have an effect on the abundance of secondary plant
products. The contents of UV protectants (flavonoids),
for instance, were significantly smaller in potato plants
overexpressing PEPC from C. glutamicum or the mutated
version of the endogenous enzyme (Häusler et al., 2001)
and were also lower in analogous transgenic tobacco
plants (Jun Li, unpublished data). This might be explained
in terms of a limitation on PEP import into the chloroplast. PEPC might compete with the initial steps of the
shikimate pathway for PEP, which serves as one of the
precursors, and thereby diminishes the flux through
the shikimate pathway. This is an hypothesis which needs
to be proven, particularly in comparison with plants overexpressing PPDK, which are capable of producing
additional PEP within the chloroplasts.
decarboxylases within the chloroplasts. The simplest
way to release CO2 would be to decarboxylate OAA by
PEPCK (Fig. 3B). This would have the advantage of
generating PEP as the substrate for PEPC without any
further enzymatic steps, but would also require (i) a
unidirectional import of OAA into the chloroplasts and
an efficient export of PEP from the chloroplasts.
For the second alternative, the decarboxylation of
malate by NADP-ME, either the unidirectional transport
of OAA or of malate would be required as malate could
either be formed from OAA by stromal NADP-MDH or
by extraplastidial NAD-MDH isozymes. Furthermore, as
NADP-ME produces pyruvate within the chloroplast, an
additional conversion into PEP by PPDK or an
alternative enzyme is necessary.
Overexpression of PEPCK
PEPCK is a cytosolic enzyme. It is therefore essential to
target it to the chloroplast by fusion to an appropriate
chloroplast targeting peptide. A cytosolic overexpression
of PEPCK together with PEPC would result in a futile
cycle. Recently, Susuki et al. have reported that an overexpression of PEPCK in rice can lead to a substantial
activity of 3 U mg 1 chlorophyll (Suzuki et al., 2000). The
PEPCK gene from Urochloa panicoides was fused to
the Rubisco small subunit (rbcS) transit sequence and
expressed under the control of the maize PEPC or PPDK
promoter. Elegant carbon isotope feeding experiments
revealed that a higher flux of 14CO2 into C4 compounds
was presumably caused by increased cytosolic PEP
levels resulting in a higher flux through PEPC. These
findings suggest that the PPT allows a significant efflux
of PEP from the chloroplast in rice plants. It also implies
that OAA can enter the chloroplast without any known
counter exchange substrate. Moreover, by feeding radiolabelled malate the same authors could show that CO2
released by the action of PEPCK enters the Calvin cycle
and causes a 3-fold increase in the labelling of 3-PGA and
sucrose compared to the controls. The rates of CO2
assimilation and the CO2 compensation point remained
unaffected. The lack of effect on photosynthesis is consistent with data obtained for transgenic tobacco plants
(Häusler et al., 2001) overexpressing the PEPCK gene
from Sinorhizobium meliloti (Osteras et al., 1995) fused to
the rbcS stromal transit sequence. However, unlike in
rice, PEPCK activity could only be detected reliably
in isolated chloroplasts of transgenic tobacco plants
(Häusler et al., 2001). The introduction of the same
construct in potato plants was not successful.
Overexpression of decarboxylating enzymes
The introduction of PEPC activity is only the first step
in establishing an intracellular CO2 pump in C3
plants. Depending on the desired mode of CO2 release
either PEPCK or NADP malic enzyme could serve as
Overexpression of chloroplastic NADP-ME
For the second approach, the increase in NADP-ME,
the cDNA of chloroplastic NADP-ME from the C3
plant Flaveria pringlei (Lipka et al., 1994) was used to
Biotechnology and C3 photosynthesis
transform potato and tobacco plants under the control of
the double CaMV 35S pomoter (Lipka et al., 1999;
Häusler et al., 2001). However, due to high activities of
endogenous cytosolic NADP-ME in solanaceous species
combined with relatively poor expression levels, the
presence of the introduced chloroplastic enzyme could
only be confirmed in chloroplast extracts (Lipka et al.,
1999). It was increased 5-fold with reference to chloroplast protein. Overexpression of the C3 chloroplast
enzyme had no large effects on photosynthetic performance of transgenic potato plants. Single NADP-ME
overexpressors of tobacco were also generated, but have
not been analysed on a physiological level.
High expression of the maize C4-type NADP-ME
in rice under the control of either the cab or CaMV
35S promoter have been reported independently by two
Japanese groups (Takeuchi et al., 2000; Tsuchida et al.,
2001). The transgenic rice plants were severely compromized in growth and exhibit photoinhibition and photodamage combined with a decline in chlorophyll contents
probably due to an excessive NADPH production within
the stroma in the light. As an indicator for the stromal
NADPHuNADP ratio the activation state of stromal
NADP-MDH (i.e. the in vivo activity as a fraction of the
activity of the fully activated enzyme) was doubled in the
transgenic lines compared to the wild type (Tsuchida
et al., 2001). Moreover, in one approach the ultrastructure of the chloroplasts appeared to be agranal, similar to
bundle-sheath chloroplasts of NADP-ME C4 plants
(Takeuchi et al., 2000). From attempts to determine C*,
a perturbation of the stromal redox states was also proposed for potato plants overexpressing NADP-ME
(Häusler et al., 2001). However, due to the relatively
low activity of the introduced NADP-ME, the transgenic
potato lines lacked any visible phenotype.
Overexpression of enzymes that regenerate PEP
from pyruvate
If CO2 is released by overexpressed NADP-malic enzyme,
the pyruvate formed has to be converted to PEP as the
substrate for PEPC. This could be achieved by overexpressing PPDK or bacterial PEP synthetase (PEPS), an
enzyme catalysing a similar reaction as PPDK but without PPi formation. As outlined earlier, PEP also serves as
a substrate for the shikimate pathway, which is localized
entirely in the chloroplast. Chloroplasts and most nongreen plastids lack the ability to produce PEP via glycolysis. Thus, PEP has to be imported from the cytosol
by the PPT. It is therefore conceivable that the
additional formation of PEP by overexpressed PPDK or
PEPS could stimulate the flux through the shikimate
pathway. In order to prevent these unpredictable sideeffects, PPDK may be overexpressed in the cytosol
(Sheriff et al., 1998).
601
Overexpression of PPDK
A successful overexpression of PPDK in C3 plants has
been reported for tobacco (Sheriff et al., 1998), potato
(Ishimaru et al., 1998) Arabidopsis (Ishimaru et al., 1997),
and rice (Nomura et al., 2000; Fukuyama et al., 2001).
Overexpression of the C4 maize PPDK gene in potato
(Ishimaru et al., 1998) resulted in about a 5-fold increase
in activity and caused a depletion in pyruvate content
and a slight elevation of PEP content. Malate content was
also considerably increased, presumably because of higher
fluxes through PEPC and subsequent malic acid formation. For rice plants, a drastic increase in PPDK activity
was achieved when the maize PPDK gene, including
introns and exons as well as its own promoter and
terminator, was used for transformation. Despite the fact
that approximately 35% of the total leaf protein consisted
of introduced PPDK, the transformants lacked a visible
phenotype or changes in growth or fertility (Fukayama
et al., 2001). Transgenic potato and tobacco plants
overexpressing the C4-type PPDK gene from Flaveria
trinervia under the control of the 35S promoter have also
been generated in the laboratory and await a thorough
analysis.
Overexpression of PEPS
As plant PPDK is highly regulated (i.e. by reversible
phosphorylation), the PEPS gene from E. coli was isolated and characterized (Niersbach et al., 1992). Potato
plants were generated overexpressing PEPS targeted to
the chloroplasts by fusion to the rbcS transit sequence
(Panstruga et al., 1997). There were no large effects on
photosynthetic CO2 assimilation and electron transport.
However, transpiration rates were slightly higher compared to the wild type and stomatal closure was delayed
appreciably indicating a perturbation in the metabolism
of stomatal guard cells (Panstruga et al., 1997). There
were some specific effects on the contents of certain
amino acids in the transgenics. The leaf contents of
glutamate were lowered by 25% whereas aspartate and
asparagine contents were increased by more than 30%
compared to the wild type. Contents of branched chain
amino acids deriving from pyruvate were also altered. The
reason for these changes has not been further elucidated
(R Panstruga et al., unpublished information). PEPS
overexpressors were compromised in shoot fresh weight,
final heights of the plants and tuber yield, and leaves
turned slightly yellowish and appeared wilted when
the plants were grown in a non-air-conditioned greenhouse during temporary hot periods in the summer
(R Panstruga et al., unpublished observations). However,
these effects were absent when plants were grown under
a controlled temperature and light regime in a growth
cabinet.
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Häusler et al.
Overexpression of the PPT
One prerequisite for an operational C4-cycle in C3 plants
is the efficient export of PEP formed in the chloroplast
by PPDK or PEPCK targeted to the chloroplast. In C3
plants there is only low activity of a chloroplastic PPT
(Fischer et al., 1997; however see also Suzuki et al., 2000).
Tobacco transformants overexpressing the heterologous
PPT from cauliflower buds under the control of the
CaMV 35S promoter have been generated and characterized in the laboratory of UI Flügge. In the transformants,
transport rates for PEP were increased 10-fold compared
to the wild type (L Voll, P Nicolay, K Fischer, RE
Häusler, UI Flügge, unpublished results).
Double transformants
The introduction of individual C4-cycle genes in C3 crops
can be regarded as a test for their functionality in the
transformed plant species. A combined overexpression of
C4-cycle genes could provide additional information on
whether or not these enzymes may work in concert. As
outlined above, single overexpression of C4-cycle genes
might perturb metabolism, as all C4 enzymes also have
distinct functions in C3 plants. The analysis of double
and multiple transformants might help to underline the
feasibility of the approach to establish a single cell C4
cycle in C3 crops.
Combined overexpression of PEPC and chloroplastic
NADP-ME
In order to release CO2 directly at the site of Rubisco,
potato double transformants with increased activities
of PEPC and chloroplastic NADP-ME from Flaveria
pringlei were generated (Lipka et al., 1999). NADP-ME
activity in isolated chloroplasts of the transformants
was increased about 5-fold compared to the wild type.
These double transformants exhibited a less pronounced
temperature-dependent decrease of CO2 assimilation
combined with a considerable decline in the electron
requirement for CO2 assimilation (i.e. the electron transport rate over apparent CO2 assimilation) at elevated
temperatures. Moreover, O2 inhibition of CO2 assimilation was attenuated significantly both at 25 8C and 35 8C
(Häusler et al., 2001). Lower rates of electron transport
for similar or even higher rates of apparent CO2 assimilation as well as an attenuation of O2 inhibition of CO2
assimilation may point at an appreciable decline in photorespiration rates. However, determinations of C* and Rd
produced equivocal results for double PEPCuNADP-ME
overexpressors (Häusler et al., 2001). C* appeared to be
decreased from 45 ml l 1 in the wild type to about
20 ml l 1 in the double transformants only at an intermediate photon flux density (PFD), but not at high light
or very low PFDs. This suggests that the NADPH
produced by chloroplastic NADP-ME might perturb
stromal redox potentials, particularly at high PFDs
(compare Tsuchida et al., 2001).
From the potato system another interesting aspect
emerged. The induction of the endogenous cytosolic
NADP-ME as a result of PEPC overexpression was
attenuated in double transformants containing PEPC
and chloroplastic NADP-ME in combination despite the
identical activities of overexpressed PEPC as compared
to the single transformants (Häusler et al., 2001). This
suggests that a redirection of the metabolic flux into the
chloroplast circumvents pleiotropic changes observed in
the single transformants and leads to the assumption that
a fully operational C4 cycle could be decoupled from the
residual metabolism in C3 plants provided that all of its
components are expressed in a well-balanced way.
Combined overexpression of PEPC with NADP-ME or
PEPCK in tobacco
A moderate overexpression of PEPC from C. glutamicum
combined with NADP-ME from F. pringlei or with
PEPCK from S. meliloti in tobacco plants had no
substantial effects on the photosynthetic performance of
the plants (Häusler et al., 2001).
Combined overexpression of PEPC with PPDK in
rice plants
Recently Ku et al. reported a 35% increase in the
photosynthetic capacity and a 22% increase in grain yield
in rice plants overexpressing PEPC and PPDK in combination (Ku et al., 2001). A detailed analysis of these
plants is awaited. It is conceivable that this observation is
linked to an increased provision of PEP by PPDK for the
shikimate pathway inside the chloroplast (Streatfield et al.,
1999; Matsuoka et al., 2000), an assumption, which
requires experimental support.
Multiple overexpression of C4-cycle enzymes
The introduction of a fully operational C4 cycle might
ultimately give the clue as to whether or not C3 photosynthesis can be improved. Multiple overexpressors
of potato have been obtained by stepwise transformation with plasmids containing different resistance genes
(kanamycin, hygromycin, sulphonylamide, and BASTA1
wbarx) for selection. Transgenic lines containing PEPC
from C. glutamicum or the mutated endogenous PEPC,
combined with NADP-ME, PPDK and the PPT have
been generated and await a thorough analysis. For
tobacco, transgenic plants overexpressing single C4-cycle
genes were genetically crossed and a number of heterozygous lines were obtained that overexpress multiple
genes (PEPC, NADP-ME, PPDK, PEPS, PEPCK, as well
as the PPT wFig. 3Bx) in various combinations (Jun Li,
unpublished results). The progenies of these plants are
Biotechnology and C3 photosynthesis
being screened at the moment for better growth in an
atmosphere containing a lowered CO2 concentration.
Species-dependent metabolic responses towards
overexpression of C4-cycle genes
Recent data (Häusler et al., 2001) show that even closely
related species such as potato and tobacco respond differentially towards an overexpression of C4-cycle genes.
For instance, the induction of cytosolic NADP-ME
in PEPC overexpressors was less apparent in tobacco
transformants compared to transgenic potato plants.
Moreover, the most pronounced attenuating effects on
photorespiration were observed with PEPCuNADP-ME
double transformants of potato plants, but for single
PEPC overexpressors of tobacco plants. It is likely that
these species-dependent differences in the acceptance for
redirecting metabolism by introduced C4-cycle enzymes
might hinder a successful introduction of an operational
C4-cycle in all of the crop plants intended to be used in
this approach.
603
space. These features of leaf anatomy are particularly
different from those of the C3 crops used in this study’s
transgenic approaches.
In an aquatic system, only diffusion of CO2 through
the water and the cell wall resistance for CO2 have to
be considered, whereas in terrestrial C3 plants diffusion
through the surrounding air, stomatal conductance for
CO2, and diffusion through the air space within the leaves
have to be taken into account as well (Farquhar et al.,
1980; Laisk and Loreto, 1996). In C4 plants the gas-tight
bundle-sheath prevents losses of CO2 from the cells and
allows high CO2 environment in the vicinity of Rubisco.
It is still an open question, whether or not the CO2
released by the introduced HCO3 uCO2 pump can be
efficiently retained within the leaf, or whether a good part
of it diffuses out of the cells into the intercellular space
and would then just result in a waste of energy (see also
Leegood, 2002). It might, for instance, become necessary
to decrease the free air space in leaves of C3 crops anduor
to minimize stomatal aperture.
What about carbonic anhydrase?
Future perspectives and possible
field applications
There has been significant progress in the overexpression
of the key enzymes of C4-type biochemistry in transgenic
C3 plants. Nevertheless, it is still uncertain whether this
approach will be sufficient to suppress photorespiration.
The following section will discuss possible alternative
anduor supplementary approaches to improve a C4-type
photosynthesis in C3 plants.
Is a C4-like CO2 pump in C3 plants realistic on
the basis of current experimental data?
So far only a limited number of transgenic plants containing a maximum of two C4-cycle enzymes have been
investigated physiologically. Most of the observed effects
were based on pleiotropic changes in metabolism. The
efficiency by which aquatic plants are capable of switching from a C3-type to a C4-like metabolism in single cells
when the availability of CO2 declines is encouraging in
the prospect that it might be possible to establish a similar
system in terrestrial C3 crops by a transgenic approach.
Moreover, the terrestrial chenopod B. aralocaspica
appears to contain a single cell CO2-concentrating
mechanism, distributed between the cytosol, the mitochondria and two types of chloroplasts in only one type
of chlorenchyma cells (Freitag and Stichler, 2000;
Vozesenskaya et al., 2001). Although this is a very exciting discovery, it ought to be considered that the anatomy
of this halophytic chenopod is adapted to semi-dry
environments with succulent leaves, central vascular
bundles, a hypodermis, large chlorenchyma cells, water
storing cells, and, most relevantly, a small intercellular air
The initial step of C4 photosynthesis is the rapid conversion of CO2 into HCO3 catalysed by carbonic
anhydrase (CA) to provide the substrate for carbon fixation by PEPC (Hatch and Burnell, 1990). It is estimated
that photosynthesis in C4 plants would be slowed down
by a factor of 104 in the absence of cytoplasmic CA
(Badger and Price, 1994). However, this aspect has not
been addressed so far in biotechnological approaches to
transfer C4-like features into C3 plants. Similar considerations as for CA might apply for pyrophosphatase and
adenylate kinase activities, which are essential for the
metabolism of the PPDK reaction products AMP and
PPi in the chloroplast. However, both enzymes are highly
active in C3 chloroplasts (Gould and Winget, 1973;
Schlattner et al., 1996).
Engineered C4 enzymes
Most approaches so far have been aimed at the introduction of the highest possible activities of the respective
enzymes in transgenic C3 plants. This is reasonable
because these enzymes are expressed to very high levels in
leaves of C4 plants. However, a high expression of the
introduced enzyme is not necessarily linked with high
in vivo activities. Modulation of enzyme activity by
covalent modification (i.e. PEPC, PPDK) and the availability of co-factors and substrates (for review see
Leegood, 1997) ought to be considered as well. For
instance, C4-specific PEPC is phosphorylated in the light
to reduce its sensitivity towards malate inhibition. It is
questionable whether the protein kinase required for
phosphorylation is expressed in the correct temporal
and spatial pattern in C3 plants. Furthermore, C4 PEPC
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Häusler et al.
has a low substrate affinity for PEP, which might be
unfavourable in the C3 environment. C3 isoforms of
PEPC show higher affinities to their respective substrates,
but are very sensitive to inhibition by malate (Svensson
et al., 1997). The catalytic properties of PEPC from
potato have been modified by genetic engineering
(T Rademacher et al., unpublished data) in the way that
the enzyme possesses both C3 (high affinity towards PEP)
and C4 (reduced sensitivity towards product inhibition)
features and is not subjected to covalent modification.
It appears to be useful and perhaps even necessary to
engineer more of the C4-cycle enzymes anduor the
respective promoters to be better adapted to the specific
requirements of a C4-like cycle in a ‘C3 environment’.
Morphological adaptations
The introduction of a ‘true C4 cycle’ into C3 plants would
ultimately require alterations in the leaf anatomy. There
are numerous deviations from the classical C3 pattern of
leaf anatomy associated with the C4 syndrome in both
mono- and dicotyledonous plants (Nelson and Langdale,
1992). However, most of these features are not absolutely
essential to perform C4 photosynthesis (Dengler and
Nelson, 1999). For example, the suberin lamella around
the bundle-sheath cells and the agranal ultrastructure of
bundle-sheath chloroplasts observed in NADP-ME type
C4 plants like maize are absent in other C4 species. Eventually, it will be crucial to separate primary carbon fixation
and photosynthetic carbon reduction in two different
tissue types or in distinct parts of one cell (as realized in
B. aralocaspica) which are in intimate contact, and to
minimize the leakage of CO2 from the tissue or the part of
the cell where Rubisco is active. Bundle-sheath cells with
distinct morphology compared to mesophyll cells are also
present in C3 plants. In Arabidopsis, reticulate mutants
have been isolated with disturbed chloroplast development in mesophyll cells, but intact chloroplasts in the
bundle-sheath indicating that the different cell types
follow a separated developmental programme (Kinsman
and Pyke, 1998). Differences in developmental programmes of mesophyll and bundle-sheath in C3 plants
might be utilized to target distinct components of the C4
cycle into the respective cell types. Moreover, the efficiency of metabolite exchange between the different cell
types would require closer vein spacing inside the leaf.
The signals determining the density of veins are not
definitely identified, but there is significant variability
within a single organism. For instance, maize foliar leaves
show a high vein density significant for C4 plants, whereas
the leaf sheath or the husk leaf exhibit distances between
the veins similar to C3 plants (Langdale et al., 1988).
Refined mutant screens and comparative gene expression
analyses will be necessary to identify the factors
responsible for changes in leaf anatomy.
Promoters
One very successful strategy is the transfer of complete genes from maize into rice (Ku et al., 1999). This
approach leads to very high expression levels and
resembles, at least in part, the spatial and light-dependent
expression patterns found in C4 plants. Overexpressed
C4-cycle enzymes might perturb the metabolism of whole
plants if transcription is not restricted to the photosynthetic tissues. The use of endogenous promoters from
C3 plants with properties similar to the C4 promoters
would be advantageous. Recently, Tsuchida et al. used
the rice cab promoter in rice for the overexpression of
maize NADP-ME, which led to a strong accumulation of
the protein in chloroplasts (Tsuchida et al., 2001).
Alternatives for leaf-specific and light-induced gene
expression include the use of other photosynthetic promoters like the lhcb promoter (Cerdan et al., 2000) or the
rbcS promoter (Kyozuka et al., 1993).
Furthermore, inducible promoters could be used (Gatz
and Lenk, 1998; Zuo and Chua, 2000). A comparison
of the induced with the non-induced state of expression
would aid physiological analysis under controlled
conditions.
Field applications
C4 plants are more productive than C3 plants when
they are grown under their respective optimum conditions
(Brown, 1999). C4 plants exhibit higher water and nitrogen use efficiencies compared to C3 plants, which results
in an increased dry matter production (reviewed in Brown,
1999). Concentrating CO2 at the site of Rubisco should
allow engineered C3 plants to reduce stomatal conductance under drought conditions without a dramatic decline
in the rate of CO2 assimilation (Drake et al., 1997). This
would allow both the use of new areas for crop
production required for feeding the growing world
population and the reduction of inputs into the system
(such as fertilizers) and thus conserving natural resources.
Both factors are much more relevant to today’s necessities
than the mere increase in biomass production.
Acknowledgements
The authors would like to thank Dr Mitsue Miyoa and Hiroshi
Fukayama for the kind provision of as yet unpublished data.
References
Andreo CS, Gonzales DH, Iglesias AA. 1987. Higher plant
phosphoenolpyruvate carboxylase: structure and regulation.
FEBS Letters 213, 1–8.
Andrews M. 1986. The partitioning of nitrate assimilation
between root and shoot of higher plants. Plant, Cell and
Environment 9, 511–519.
Biotechnology and C3 photosynthesis
Aoyagi K, Bassham JA. 1984. Pyruvate orthophosphate dikinase
mRNA organ specificity in wheat and maize plants. Plant
Physiology 76, 278–280.
Arp WJ, Van Mierlo JEM, Berendse FEM, Snijders W. 1998.
Interaction between elevated CO2 concentration, nitrogen and
water: effects on growth and water use of six perennial plant
species. Plant, Cell and Environment 21, 1–11.
Asai N, Nakajima N, Tamaoki M, Kamada H, Kondo N. 2000.
Role of malate synthesis mediated by phosphoenolpyruvate
carboxylase in guard cells in the regulation of stomatal
movement. Plant Cell Physiology 41, 10–15.
Badger MR, Price GD. 1994. The role of carbonic anhydrase in
photosynthesis. Annual Review of Plant Physiology and Plant
Molecular Biology 45, 369–392.
Benfey PN, Chua NH. 1990. The Cauliflower Mosaic Virus 35S
promoter combinatorial regulation of transcription in plants.
Science 250, 959–966.
Besford RT. 1990. The greenhouse effects on the acclimation
of tomato plants growing in high carbon dioxide relative to
changes in Calvin cycle enzymes. Journal of Plant Physiology
136, 458–463.
Brooks A, Farquhar GD. 1985. Effect of temperature on the
CO2uO2 specificity of ribulose-1,5-bisphosphate carboxylaseu
oxygenase and the rate of respiration in the light. Planta 165,
397–406.
Brown RH. 1999. Agronomic implications of C4 photosynthesis.
In: Sage RF, Monson RK, eds. C4 plant biology. Academic
Press, 473–507.
Casati P, Drincovich MF, Edwards GE, Andreo CS. 1999.
Minireview. Malate metabolism in plant defence. Photosynthesis Research 61, 99–105.
Casati P, Lara MV, Andreo CS. 2000. Induction of a C4-like
mechanism of CO2 fixation in Egeria densa, a submerged
aquatic species. Plant Physiology 123, 1611–1621.
Cerdan PD, Staneloni RJ, Ortega J, Bunge MM,
Rodriguez-Batiller MJ, Sanchez RA, Casal JJ. 2000.
Sustained but not transient phytochrome A signaling targets
a region of an lhcb1*2 promoter not necessary for phytochrome B action. The Plant Cell 12, 1203–1211.
Chen K, Hu G, Kreutgen N, Lenz F. 1997. The effect of
CO2 concentration on strawberries. I. Plant growth analysis.
Journal of Applied Botany 71, 168–172.
Chen R. 1998. Plant NADP-dependent isocitrate dehydrogenases are predominantly localized in the cytosol. Planta
207, 280–285.
Chen Z, Spreitzer RJ. 1992. How various factors influence
the CO2uO2 specificity of ribulose-1,5 bisphosphate
carboxylaseuoxygenase. Photosynthesis Research 31, 157–164.
Chollet R, Vidal J, O’Leary MH. 1996. Phosphoenolpyruvate
carboxylase: a ubiquitous, highly regulated enzyme in plants.
Annual Review of Plant Physiology and Plant Molecular
Biology 47, 273–298.
Cockburn W, Ting IP, Sternberg LO. 1979. Relationships
between stomatal behaviour and internal carbon dioxide
concentration in crassulacean acid metabolism plants. Plant
Physiology 63, 1029–1032.
Cooper TG, Wood HG. 1971. The carboxylation of phosphoenolpyruvate and pyruvate. II. The active species of
‘CO2’ utilized by phosphoenolpyruvate carboxylase and
pyruvate carboxylase. Journal of Biological Chemistry
246, 5488–5490.
de Groote D, Kennedy RA. 1977. Photosynthesis in Elodea
canadensis Michx. Plant Physiology 59, 1133–1135.
Dengler NG, Nelson T. 1999. Leaf structure and development in
C4 plants. In: Sage RF, Monson RK, eds. C4 plant biology.
San Diego: Academic Press, 133–172.
605
Douce R, Neuburger M. 1989. The uniqueness of plant
mitochondria. Annual Review of Plant Physiology and Plant
Molecular Biology 40, 371–414.
Drake BG, Gonzalez-Meler MA, Long SP. 1997. More efficient
plants: a consequence of rising atmospheric CO2? Annual
Review of Plant Physiology and Plant Molecular Biology
48, 609–639.
Duff SMG, Chollet R. 1995. In vivo regulation of wheat-leaf
phosphoenolpyruvate carboxylase by reversible phosphorylation. Plant Physiology 107, 775–782.
Edwards GE, Andreo CS. 1992. NADP-malic enzymes from
plants. Photochemistry 31, 1845–1857.
Edwards GE, Furbank RT, Hatch MD, Osmond CB. 2001. What
does it take to be C4? Lessons from the evolution of C4
photosynthesis. Plant Physiology 125, 36–49.
Farquhar GD, von Caemmerer S, Berry JA. 1980. A biochemical
model of photosynthetic CO2 assimilation in leaves of C3
species. Planta 149, 78–90.
Fischer K, Kammerer B, Gutensohn M, Arbinger B, Weber A,
Häusler RE, Flügge UI. 1997. A new class of plastidic
phosphate translocators: a putative link between primary and
secondary metabolism by the phosphoenolpyruvateu
phosphate antiporter. The Plant Cell 9, 453–462.
Flügge UI, Stitt M, Heldt HW. 1985. Light-driven uptake of
pyruvate into mesophyll chloroplasts from maize. FEBS
Letters 183, 335–339.
Freitag H, Stichler W. 2000. A remarkable new leaf type with
unusual photosynthetic tissue in a central Asiatic genus of
Chenopodiacea. Plant Biology 2, 154–160.
Fukayama H, Tsuchida H, Agarie S et al. 2001.
Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiology
127, 1136–1146.
Gatz C, Lenk I. 1998. Promoters that respond to chemical
inducers. Trends in Plant Science 3, 352–358.
Gehlen J, Panstruga R, Smets H, Merkelbach S, Kleines M,
Porsch P, Fladung, Becker I, Rademacher T, Häusler RE,
Hirsch H-J. 1996. Effects of altered phosphoenolpyruvate
carboxylase activities on transgenic C3 plant Solanum
tuberosum. Plant Molecular Biology 32, 831–848.
Gould JM, Winget GD. 1973. A membrane-bound alkaline
inorganic pyrophosphatase in isolated spinach chloroplasts.
Archives of Biochemistry and Biophysics 154, 606–613.
Hatch MD, Burnell JN. 1990. Carbonic anhydrase activity in
leaves and its role in the first step of C4 photosynthesis. Plant
Physiology 93, 825–828.
Hatch MD, Osmond CB. 1976. Compartmentation and transport in C4 photosynthesis. In: Stocking CR, Heber U, eds.
Encyclopedia of plant physiology, New series, Vol. 3. Berlin:
Springer Verlag, 144–184.
Häusler RE, Kleines M, Uhrig H, Hirsch H-J, Smets H. 1999.
Overexpression of phosphoenolpyruvate carboxylase from
Corynebacterium glutamicum lowers the CO2 compensation
point (C*) and enhances dark and light respiration in
transgenic potato. Journal of Experimental Botany 336,
1231–1242.
Häusler RE, Rademacher T, Li J, Lipka V, Fischer KL,
Schubert S, Kreuzaler F, Hirsch H-J. 2001. Single and double
overexpression of C4-cycle genes had differential effects on the
pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato
and tobacco plants. Journal of Experimental Botany 52,
1785–1803.
Holaday AS, Bowes G. 1980. C4 acid metabolism and dark
CO2 fixation in a submerged aquatic macrophyte (Hydrilla
verticillata). Plant Physiology 65, 331–335.
606
Häusler et al.
Hudspeth RL, Grula JW, Dai Z, Edwards GE, Ku MSB. 1992.
Expression of maize phosphoenolpyruvate carboxylase in
transgenic tobacco. Plant Physiology 98, 458–464.
Ishimaru K, Ishikawa I, Matsuoka M, Ohsugi R. 1997. Analysis
of a C4 maize pyruvate, orthophosphate dikinase expressed in
C3 transgenic Arabidopsis plants. Plant Science 129, 57–64.
Ishimaru K, Okawa Y, Ishige T, Tobias DJ, Ohsugi R. 1998.
Elevated pyruvate, orthophosphate dikinase (PPDK)
activity alters carbon metabolism in C3 transgenic potatoes
with a C4 maize PPDK gene. Physiolgia Plantarum 103,
340–346.
Izui K, Ishijima S, Yamagushi Y, Katagiri F, Murata T,
Shigesada K, Sugiyama T, Katsuki H. 1986. Cloning and
sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway of maize. Nucleic
Acid Research 14, 1615–1628.
Jordan DB, Ogren WL. 1984. The CO2uO2 specificity of ribulose
1,5-bisphosphate carboxylaseuoxygenase. Dependence on
ribulose bisphosphate concentration, pH and temperature.
Planta 161, 308–313.
Kellogg EA. 1999. Phylogenetic aspects of the evolution of C4
photosynthesis. In: Sage RF, Monson RK, eds. C4 plant
biology. London: Academic Press, 411–443.
Keys AJ, Bird IF, Cornelius MJ, Lea PJ, Wallsgrove RM,
Miflin BJ. 1978. Photorespiratory nitrogen cycle. Nature 275,
741–743.
Kinsman EA, Pyke KA. 1998. Bundle sheath cells and cellspecific plastid development in Arabidopsis leaves.
Development 125, 1815–1822.
Knee M, Finger FL, Lagrimini M. 1996. Evidence for a cytosolic
NADP-malic enzyme in tomato. Phytochemistry 42, 11–16.
Kogami H, Shono M, Koike T, Yanagisawa S, Izui K, Sentoku N,
Tanifuji S, Uchimiya H, Toki S. 1994. Molecular and
physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the
control of the cauliflower mosaic virus 35S promoter.
Transgenic Research 3, 287–296.
Ku MSB, Agarie S, Nomura M, Fukayama H, Tsuchida H,
Ono K, Hirose S, Toki S, Miyao M, Matsuoka M. 1999. Highlevel expression of maize phosphoenolpyruvate carboxylase in
transgenic rice plants. Nature Biotechnology 17, 76–80.
Ku MSB, Cho DH, Li X, Jiao DM, Pinto M, Miyao M,
Matsuoka M. 2001. Introduction of genes encoding C4
photosynthesis enzymes into rice plants: physiological consequences. Rice Biotechnology: Improving Yield, Stress
Tolerance and Grain Quality 236, 100–116.
Ku MSB, Kano-Murakami Y, Matsuoka M. 1996. Evolution and
expression of C4 photosynthesis gene. Plant Physiology 111,
949–957.
Kyozuka J, McElroy D, Hayakawa T, Xie Y, Wu R,
Shimamoto K. 1993. Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in
transgenic rice. Plant Physiology 102, 991–1000.
Laisk A, Loreto F. 1996. Determining photosynthetic parameters from leaf CO2 exchange and chlorophyll fluorescence.
Ribulose-1,5-bisphosphate carboxylaseuoxygenase specificity
factor, dark respiration in the light, excitation distribution
between photosystems, alternative electron transport rates
and mesophyll diffusion resistance. Plant Physiology 110,
903–912.
Langdale JA, Zelitch I, Miller E, Nelson T. 1988. Cell position
and light influence C4 versus C3 patterns of photosynthetic
gene expression in maize. EMBO Journal 7, 3643–3651.
Latzko E, Kelly GJ. 1983. The many-faceted function of
phosphoenolpyruvate carboxylase in C3 plants. Physiologia
Ve´ge´tale 21, 805–815.
Leegood RC, Acheson RM, Tecsi LI, Walker RP. 1999. The
many-faceted function of phosphoenolpyruvate carboxykinase in plants. In: Kruger NJ, Hill SA, Ratcliffe RG, eds.
Regulation of primary metabolism in plants. Dodrecht:
Kluwer, 37–57.
Leegood RC. 1997. The regulation of C4 photosynthesis.
Advances in Botanical Research 26, 251–316.
Leegood RC. 2002. C4 photosynthesis: principles of CO2 concentration and prospects for its introduction into C3 plants.
Journal of Experimental Botany 53, 581–590.
Lipka B, Steinmüller K, Rosche E, Börsch D, Westhoff P. 1994.
The C3 plant Flaveria pringlei contains a plastidic
NADP-malic enzyme which is orthologous to the C4 isoform
of the C4 plant F. trinervia. Plant Molecular Biology 26,
1775–1783.
Lipka V, Häusler RE, Rademacher T, Li J, Hirsch H-J,
Kreuzaler F. 1999. Solanum tuberosum double transgenic
expressing phosphoenolpyruvate carboxylase and NADPmalic enzyme display reduced electron requirement for CO2
fixation. Plant Science 144, 93–105.
Magnin NC, Cooley BA, Reiskind JB, Bowes G. 1997.
Regulation and localization of key enzymes during the
induction of Kranz-less, C4-type photosynthesis in Hydrilla
verticillata. Plant Physiology 115, 1681–1689.
Martinoia E, Rentsch D. 1994. Malate compartmentation—
responses to a complex metabolism. Annual Review of Plant
Physiology and Plant Molecular Biology 45, 447–467.
Matsuoka M, Fukayama H, Tsuchida H, Nomura M, Agari S,
Ku MSB, Miyao M. 2000. How to express some C4
photosynthesis genes at high levels in rice. In: Sheehy JE,
Mitchell PL, Hardy B. eds. Redesigning rice photosynthesis to
increase yield. Proceedings of the workshop on The Quest
to Reduce Hunger: Redesigning rice photosynthesis, 30
November to 3 December 1999, Los Banos, Phillipines.
International Rice Research Institute and Amsterdam:
Elsevier Science BV, 167–175.
Matsuoka M, Furbank RT, Fukayama H, Miyao M. 2001.
Molecular engineering of C4 photosynthesis. Annual Review of
Plant Physiology and Plant Molecular Biology 52, 297–314.
Melzer E, O’Leary MH. 1987. Anaplerotic CO2 fixation by
phosphoenolpyruvate carboxylase in C3 plants. Plant
Physiology 84, 58–60.
Merkelbach S, Gehlen J, Denecke M, Hirsch HJ, Kreuzaler F.
1993. Cloning, sequence analysis and expression of a cDNA
encoding active phosphoenolpyruvate carboxylase of the
C3 plant Solanum tuberosum. Plant Molecular Biology 23,
881–888.
Nelson T, Langdale JA. 1992. Developmental genetics of C4
photosynthesis. Annual Review of Plant Physiology and Plant
Molecular Biology 43, 25–47.
Niersbach M, Kreuzaler F, Geerse RH, Postma PW, Hirsch HJ.
1992. Cloning and nucleotide sequence of the Escherichia coli
K-12 ppsA gene, encoding PEP synthase. Molecular Genetics
and Genomics 231, 332–336.
Nomura M, Sentoku N, Tajima S, Matsuoka M. 2000.
Expression patterns of cytoplasmic pyruvate, orthophosphate
dikinase of rice (C3) and maize (C4) in a C3 plant, rice.
Australian Journal of Plant Physiology 27, 343–347.
O’Leary MH. 1982. Phosphoenolpyruvate carboxylase: an
enzymologist’s view. Annual Review of Plant Physiology
33, 297–315.
Ogren WL. 1984. Photorespiration: pathways, regulation and
modification. Annual Review of Plant Physiology 35, 415–442.
Oliver DJ. 1994. The glycine decarboxylase complex from plant
mitochondria. Annual Review of Plant Physiology and Plant
Molecular Biology 45, 323–337.
Biotechnology and C3 photosynthesis
Osmond CB. 1978. Crassulacean acid metabolism, a curiosity in
context. Annual Review of Plant Physiology 29, 379–414.
Osteras M, Driscoll BT, Finan TM. 1995. Molecular and
expression analysis of the Rhizobium meliloti phosphoenol
pyruvate carboxykinase ( pckA) gene. Journal of Bacteriology
177, 1452–1460.
Panstruga R, Hippe-Sanwald S, Lee Y-K, Lataster M, Lipka V,
Fischer R, Liao YC, Häusler RE, Kreuzaler F, Hirsch HJ.
1997. Expression and chloroplast-targeting of active phophoenolpyruvate synthetase from Escherichia coli in Solanum
tuberosum. Plant Science 127, 191–205.
Poetsch W, Hermans J, Westhoff P. 1991. Multiple cDNAs of
phosphoenolpyruvate carboxylase in the C4 dicot Flaveria
trinervia. FEBS Letters 292, 133–136.
Polle A. 1996. Mehler reaction: friend or foe in photosynthesis?
Botatica Acta 109, 84–89.
Raschke K, Hedrich R, Reckmann U, Schroeder JI. 1988.
Exploring biophysical and biochemical components of the
osmotic motor that drives stomatal movement. Botanica Acta
101, 283–294.
Reiskind JB, Madsen TV, van Ginkel LC, Bowes G. 1997.
Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submerged monocot. Plant, Cell and Environment 20, 211–220.
Sage RF, Pearcy RW. 1987. The nitrogen use efficiency of C3 and
C4 plants. I. Leaf nitrogen, growth and biomass partitioning
in Chenopodium album (L.) and Amaranthus retroflexus (L.).
Plant Physiology 84, 954–958.
Salvucci ME, Bowes G. 1981. Induction of reduced photorespiratory activity in submerged and amphibious aquatic
macrophytes. Plant Physiology 67, 335–340.
Salvucci ME, Bowes G. 1983. Two photosynthetic mechanisms
mediating the low photorespiratory state in submerged
aquatic angiosperm. Plant Physiology 73, 488–496.
Schaaf J, Walter MH, Hess D. 1995. Primary metabolism in
plant defense. Regulation of bean malic enzyme gene
promoter in transgenic tobacco by developmental and
environmental cues. Plant Physiology 108, 949–960.
Scheibe R. 1987. NADPq-malate dehydrogenase in C3 plants:
regulation and role of a light-activated enzyme. Physiologia
Plantarum 71, 393–400.
Schlattner U, Wagner E, Greppin H, Bonzon M. 1996.
Chloroplast adenylate kinase from tobacco: purification and
partial characterization. Phytochemistry 42, 589–594.
Schmid J, Amrhein N. 1995. Molecular organization of the
shikimate pathway in higher plants. Phytochemistry 39,
737–749.
Schnabl H. 1981. The compartmentation of carboxylating and
decarboxylating enzymes in guard cell protoplasts. Planta
152, 307–313.
Sheriff A, Meyer H, Riedel E, Schmitt JM, Lapke C. 1998. The
influence of plant pyruvate, orthophosphate dikinase on a C3
plant with respect to the intracellular location of the enzyme.
Plant Science 136, 43–57.
607
Spalding MH, Stumpf DK, Ku MSB, Burris RH, Edwards GE.
1979. Crassulacean acid metabolism and diurnal variations of
internal CO2 and O2 concentrations in Sedum praealtum DC.
Australien Journal of Plant Physiology 6, 557–567.
Spencer WE, Wetzel RG, Teeri J. 1996. Photosynthetic
phenotype plasticity and the role of phosphoenolpyruvate
carboxylase in Hydrilla verticillata. Plant Science 118, 1–9.
Streatfield SJ, Weber A, Kinsman EA, Häusler RE, Li J,
Post-Beitenmiller D, Kaiser WM, Pyke KA, Flügge UI,
Chory J. 1999. The phosphoenolpyruvateuphosphate translocator is required for phenolic metabolism, palisade cell
development and plastid-dependent nuclear gene expression.
The Plant Cell 11, 1609–1621.
Suzuki S, Murai N, Burnell J, Arai M. 2000. Changes in
photosynthetic carbon flow in transgenic rice plants that
express C4-type phosphoenolpyruvate carboxykinase from
Urochloa panicoides. Plant Physiology 124, 163–172.
Svensson P, Blasing OE, Westhoff P. 1997. Evolution of the
enzymatic characteristics of C4 phosphoenolpyruvate carboxylase—a comparison of the orthologous ppcA phosphoenolpyruvate carboxylases of Flaveria trinervia (C4) and
Flaveria pringlei (C3). European Journal of Biochemistry 246,
452– 460.
Takeuchi Y, Akagi H, Kamasawa N, Osumi M, Honda H. 2000.
Aberrant chloroplasts in transgenic rice plants expressing a
high level of maize NADP-dependent malic enzyme. Planta
211, 265–274.
Teramura AH, Sullivan JH, Ziska LH. 1990. Interaction of
elevated UV-B radiation and carbon dioxide on productivity
and photosynthetic characteristics in wheat, rice and soybean.
Plant Physiology 94, 470–475.
Tsuchida H, Tamai T, Fukayama H, Agarie S, Nomura M,
Onodera H, Ono K, Nishizawa Y, Lee B-H, Hirose S, Toki S,
Ku MSB, Matsuoka M, Miyao M. 2001. High level expression
of C4-specific NADP-malic enzyme in leaves and impairment
of photoautotrophic growth in a C3 plant, rice. Molecular
engineering of C4 photosynthesis. Plant Cell Physiology
42, 138–145.
van Ginkel LC, Bowes G, Reiskind JB, Prins HBA. 2001. A
CO2-flux mechanism operating via pH-polarity in Hydrilla
verticillata leaves with C3 and C4 photosynthesis.
Photosynthesis Research 68, 81–88.
Van Quy L, Lamaze T, Champigny M-L. 1991. Short-term
effects of nitrate on sucrose synthesis in wheat leaves. Planta
185, 53–57.
Voznesenskaya EV, Franceschi VR, Kiirats O, Freitag H,
Edwards GE. 2001. Kranz anatomy is not essential for
terrestrial C4 plant photosynthesis. Nature 414, 543–546.
Zhang XQ, Li B, Chollet R. 1995. In vivo regulatory phosphorylation of soybean nodule phosphoenolpyruvate carboxylase. Plant Physiology 108, 1561–1568.
Zuo J, Chua N-H. 2000. Chemical-inducible systems for
regulated expression of plant genes. Current Opinion in
Biotechnology 11, 146–151.