Journal of Experimental Botany, Vol. 53, No. 369, pp. 581–590, April 2002
REVIEW ARTICLE
C4 photosynthesis: principles of CO2 concentration
and prospects for its introduction into C3 plants
Richard C. Leegood1
Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield,
Sheffield S10 2TN, UK
Received 31 October 2001; Accepted 13 December 2001
Abstract
C4 photosynthesis has a number of distinct properties that enable the capture of CO2 and its concentration in the vicinity of Rubisco, so as to reduce the
oxygenase activity of Rubisco, and hence the rate of
photorespiration. The aim of this review is to discuss
the properties of this CO2-concentrating mechanism,
and thus to indicate the minimum requirements of
any genetically-engineered system. In particular,
the Kranz leaf anatomy of C4 photosynthesis and
the division of the C4-cycle between two cell types
involves intercellular co-operation that requires modifications in regulation and transport to make C4
photosynthesis work. Some examples of these modifications are discussed. Comparisons are made with
the C4-type photosynthesis found in single-celled
C4-type CO2-concentrating mechanisms, such as
that found in the aquatic plant, Hydrilla verticillata
and the single-celled C4 system found in the terrestrial chenopod Borszczowia aralocaspica. The outcome of recent attempts to engineer C4 enzymes
into C3 plants is discussed.
Key words: C4 photosynthesis, CO2 capture, CO2 concentration, photorespiration, Rubisco.
Introduction
The discovery of C4 photosynthesis in the 1960s and
awareness of the high photosynthetic capacity of C4, as
compared with C3, plants created an upsurge of interest in
the impact of photorespiration on the carbon economy of
C3 plants, its origins in the Rubisco oxygenase reaction,
and in the ecophysiological relationships between C3 and
1
Fax: q44 (0) 114 222 0050. E-mail: r.leegood@shef.ac.uk
ß Society for Experimental Biology 2002
C4 plants. This was recorded strikingly in the proceedings
of a conference held in Canberra in 1970 (Hatch et al.,
1971). Later, Burris and Black wrote, ‘A continuing
interest in plant productivity has been quickened in recent
years primarily by a growing concern for feeding a rapid
expanding world population and by the discovery of
the C4 pathway of photosynthetic carbon assimilation.
Population growth has served as a goad to research, and
the low photorespiration and high efficiency of the C4
pathway have suggested a promising route to aid in
achieving increased plant yields’ (Burris and Black, 1976).
At the time, attempts were made to transfer the advantages of C4 photosynthesis (reduced photorespiration,
and increased nitrogen, water efficiency and, under certain
conditions, increased light use efficiency) into C3 plants
by breeding. Although C3 and C4 species of Atriplex
were hybridized, the independent inheritance of the genes
conferring C4 characteristics, such as Kranz anatomy,
elevated PEP carboxylase and low CO2 compensation
point, indicated that plant breeding was unlikely to be a
successful route (Björkman et al., 1971; Björkman, 1976).
However, the advent of genetic engineering in crop plants
has recently resulted in attempts to introduce C4 characteristics into C3 plants, such as tobacco, potato and
rice, by genetic manipulation (Matsuoka et al., 2001).
Clearly, it is easier to over-express enzymes of the C4
pathway either individually or in concert, than it is
to introduce the complex Kranz leaf anatomy of C4
photosynthesis, with its division of labour between
thin-walled mesophyll cells surrounding the thick-walled
chlorenchymatous bundle-sheath. For this reason, considerable interest has focused on engineering single-celled
C4-type CO2-concentrating mechanisms, such as that
found in the aquatic plant, Hydrilla verticillata. The
aim of this review is to discuss the requirements of
a CO2-mechanism as found in C4 plants, and thus to
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Leegood
indicate the minimum requirements of any geneticallyengineered system.
What are the requirements for a
CO2-concentrating mechanism in C4 plants?
C4 photosynthesis has a number of features characteristic
of, and essential to, CO2-concentrating mechanisms
in general. These components have been defined earlier
(Badger and Spalding, 2000) (Fig. 1). The number for
each component discussed below corresponds to those
in Fig. 1.
(1) An active, photosynthetically-driven,
CO2 capture system
In C4 plants this role is taken by PEP carboxylase
operating in the cytosol of the specialized mesophyll
cells. A specialized C4 isoform of PEP carboxylase is
expressed in very high amounts (c. 20 times that of the
housekeeping ‘C3’ isoform), under the control of a promoter that directs expression specifically in the mesophyll
cells (Stockhaus et al., 1997). The properties of the C4
PEP carboxylase are slightly modified compared with
the C3 form (Chollet et al., 1996). Mutants that lack
the C4 form, but which retain the housekeeping form of
PEP carboxylase, are unable either to concentrate CO2 or
to assimilate CO2 diffusing from the atmosphere directly
in the bundle-sheath (Dever et al., 1995).
An aspect which is of crucial importance, but which is
frequently overlooked, is the role of carbonic anhydrase
in C4 photosynthesis. PEP carboxylase utilizes bicarbonate, rather than CO2. This means that CO2 entering the
mesophyll cells from the atmosphere must be rapidly converted to bicarbonate, catalysed by carbonic anhydrase
(Hatch and Burnell, 1990; Badger and Price, 1994).
Carbonic anhydrase in the leaves of C4 plants is probably
confined to the mesophyll cytosol, albeit at activities
Fig. 1. A generalized model of the components of a CO2-concentrating
mechanism. Components 1 to 7 represent the generic components of
a CCM as indicated in the text headings. (Adapted from Badger and
Spalding, 2000.)
which are only just sufficient to ensure that the conversion
of CO2 to bicarbonate does not limit photosynthesis. This
is largely attributable to the low concentration of CO2 in
the mesophyll (;4 mM) compared with the high Km(CO2)
of carbonic anhydrase (between 0.8 and 2.8 mM in a
range of C4 species) (Hatch and Burnell, 1990). In C3
plants, on the other hand, carbonic anhydrase is largely
confined to the chloroplast (Badger and Price, 1994).
(2) A supply of photosynthetic energy
In C4 plants ATP is used to drive the C4 cycle. In
the simplest form of C4 photosynthesis, found in NADPmalic enzyme species, such as sugar cane (Fig. 2),
photosynthetically-generated ATP (2ATP) is used to
regenerate PEP from pyruvate, catalysed by pyruvate,
Pi dikinase, and 1 NADPH is used to reduce oxaloacetate
to malate in the mesophyll. In the bundle-sheath chloroplast 1 NADPH is generated by the action of NADPmalic enzyme. The C4 cycle is thus an ATP-driven CO2
pump, utilizing 2ATP per CO2 transferred from the
mesophyll to the bundle-sheath.
(3) An intermediate pool of captured CO2
Oxaloacetate, the immediate product of PEP carboxylation is highly reactive and does not accumulate to
high concentrations in maize (an NADP-malic enzyme
species), though it is present in significant quantities
Fig. 2. The CO2-concentrating mechanism in an NADP-malic enzyme
type C4 plant, sugar cane. PEP carboxylase (PEPC) in the mesophyll
cytosol fixes bicarbonate, generated from CO2 by carbonic anhydrase
(CA) and produces oxaloacetate (OAA). OAA is reduced to
malate in the mesophyll chloroplast by NADP-malate dehydrogenase
(NADP-MDH). Malate is transported to the bundle-sheath via the
plasmodesmata and is decarboxylated in the bundle-sheath chloroplast
by NADP-malic enzyme (NADP-ME). The released CO2 is fixed by
Rubisco in the Benson–Calvin cycle. PEP is regenerated from
pyruvate by pyruvate, Pi dikinase (PPDK) in the mesophyll chloroplast
(the reaction is shown as the combined actions of PPDK and
adenylate kinase). In addition, glycerate-3-P from the Benson–Calvin
cycle is shuttled to the mesophyll for reduction to triose-P. Membrane
transporters are indicated by filled circles.
C4 photosynthesis
in Amaranthus edulis, an NAD-malic enzyme species
(Leegood and von Caemmerer, 1988). Generally oxaloacetate is converted to C4 acids, such as malate and
aspartate, which act as transient stores of fixed CO2
(Hatch, 1971). The relative proportions of malate and
aspartate formed from oxaloacetate are dependent on
the C4 sub-type (little aspartate is formed in pure
NADP-malic enzyme species), and probably other factors
such as light intensity and nitrogen nutrition (Khamis
et al., 1992).
(4) A mechanism to release CO2 from the
intermediate pool
In C4 plants this role is taken by specific enzymes
decarboxylating C4 acids in the bundle-sheath. These
decarboxylases (NAD- and NADP-malic enzymes and
PEP carboxykinase) all release CO2 (the substrate of
Rubisco) rather than bicarbonate, although this release
occurs in the chloroplast in the case of NADP-malic
enzyme, in the cytosol in the case of PEPCK, and in
the mitochondria in the case of NAD-malic enzyme.
Although C4 plants have traditionally been classified into
three subgroups based on the presence of one of these
decarboxylases, it is now becoming apparent that there
is much more diversity. In PEPCK types, PEPCK works
in tandem with NAD-malic enzyme (Burnell and Hatch,
1988b). Many NADP-malic enzyme species, including
maize, also have PEPCK as an auxiliary decarboxylase,
decarboxylating aspartate (Gutierrez et al., 1974; Wingler
et al., 1999), and other NADP-malic enzyme types exhibit
appreciable decarboxylation of aspartate as well as malate
(Meister et al., 1996). This diversity probably reflects
the multiple evolutionary origins of C4 photosynthesis.
The advantages and disadvantages of particular pathways
of decarboxylation are unclear, but their presence is a
testament to the flexibility of plant metabolism.
(5) A compartment in which to concentrate
CO2 around Rubisco
In the majority of C4 plants the photosynthetic cells
within the leaf are organized in two concentric cylinders.
The outer cylinder comprises thin-walled mesophyll cells
with large intercellular spaces which radiate from the inner
cylinder of thick-walled bundle sheath cells. The thickened cell wall is a major barrier to the diffusion of solutes
and gases in all C4 plants and the bundle-sheath is
the compartment in which CO2 is concentrated. Direct
measurements of the inorganic carbon pool show that it is
about some 10–20 times the mesophyll CO2 concentration
(Hatch, 1999). Although many C3 plants possess a bundlesheath, it is seldom green to the degree found in C4 plants.
In rice or barley, for example, the bundle-sheath contains
only a few chloroplasts. In most C3 plants, the parenchymatous bundle-sheath may be important in the loading
583
of assimilates into the vasculature (Koroleva et al., 2000)
rather than playing any major role in CO2 fixation. It has
also been suggested that increased bundle-sheath size may
be important in maintaining hydraulic integrity in hot,
arid environments (Sage, 2001) and could, therefore, be
preadaptive. Factors that are involved in the development
of the bundle-sheath have yet to be elucidated at the
molecular level (Taylor, 2000; Dengler and Nelson, 1999).
(6) A means to reduce leakage of CO2 from the
site of CO2 elevation
Three factors are important here. First, the C4 structure
provides a long liquid diffusion pathway from the bundlesheath organelles to the intercellular spaces surrounding
the mesophyll cells, particularly if the chloroplasts
are centripetally arranged in the bundle-sheath, as, for
example, in NAD-malic enzyme and PEP carboxykinase
type C4 species. Second, the bundle sheath has thickened
walls, which reduce its permeability to CO2 (Table 1). One
factor that may affect leakage of CO2 from the bundlesheath is the occurrence of a suberized lamella. It is
absent in dicotyledonous species, and in grasses is present
only in species with either an uneven bundle sheath outline or with centrifugally located chloroplasts. In those
species with uneven cell outlines the suberized lamella
may be important in restricting CO2 leakage through the
high surface area of the bundle sheathumesophyll interface (Hattersley, 1992). Third, the absence of carbonic
anhydrase in bundle sheath cells is necessary for the
effective concentration of CO2 in the bundle-sheath. This
is critically important because the substrate for Rubisco is
CO2, not bicarbonate, and carbonic anhydrase activity
would lead to the accumulation of bicarbonate rather
than CO2 as the predominant inorganic carbon species.
For example, at equilibrium at pH 8 the bicarbonate
concentration would be 50 times that of CO2 (Furbank
and Hatch, 1987; Burnell and Hatch, 1988a).
Detailed modelling of the compartmentation of the
inorganic carbon pool in mesophyll and bundle sheath
has shown that the efflux of HCO3 via plasmodesmata is
insignificant compared to the flux of C4 acids (Furbank
and Hatch, 1987). It has, therefore, been suggested that
Table 1. Permeability coefficients for CO2 (PCO2) in different
structures
Structure
Artificial lipid bilayer
Xanthium leaf cells
C4 bundle-sheath cells
Chlamydomonas plasma
membrane
Cyanobacterial
carboxysome
PCO2 (cm s 1)
Reference
1
3.5 3 10
0.7 to 1.7 3 10 1
1.6 to 4.5 3 10 3
0.76 to 1.49 3 10
10
4
3
Gutknecht et al., 1988
Evans et al., 1986
Furbank et al., 1989
Sültemeyer and Rinast,
1996
Badger and Spalding,
2000
584
Leegood
leakage of HCO3 via the plasmodesmata is not likely to
be a serious problem, nor is the leakage of CO2, because
the diffusion coefficients of gases in solution are 104 times
less than in air. Of course, if substantial CO2 leakage
occurred, this would reduce the efficiency of C4 photosynthesis by acting as a futile cycle and increasing its
quantum (light) requirement (Hatch et al., 1995).
(7) Modification of the kinetic properties of Rubisco
In C4 plants, Rubisco has an intermediate affinity
for CO2. The Km(CO2) of C4 grasses ranges between
28–60 mM, compared to C3 grasses with a range of
13–26 mM (Yeoh et al., 1980), a high Srel (the relative
specificity factor for Rubisco, relating the carboxylase to
oxygenase reaction kinetics) comparable to that in C3
plants, and a catalytic turnover rate about double that of
C3 Rubisco (Seemann et al., 1984). An increased catalytic
turnover of Rubisco is always associated with a reduced
affinity for CO2, i.e. there is a trade-off between the two
(von Caemmerer and Quick, 2000; Badger and Spalding,
2000). Since Rubisco in C4 plants operates at high CO2
concentrations (well above the Km for CO2), the effects of
the reduced affinity are negligible, and the increased
catalytic turnover results in improved nitrogen use
efficiency in that C4 plants maintain a higher photosynthetic rate for a lower investment in Rubisco, which
can form up to 50% of the soluble protein in the leaves
of C3 plants.
Specific requirements of C4 photosynthesis
In addition to these general requirements of a CO2concentrating mechanism, C4 plants, by virtue of having
the C4 cycle separated into two distinct cell types,
have specialized arrangements for both intracellular and
intercellular transport of metabolites, and differences in
the regulation, that are crucial to its operation.
One vital aspect of C4 photosynthesis is metabolite
movement between the bundle sheath and the mesophyll.
This is sustained by diffusion via numerous plasmodesmata, driven by gradients in their concentrations
(Leegood, 1985; Stitt and Heldt, 1985). The C4 plasmodesmatal frequency can be 3–5 times the C3 frequency at
the mesophyllubundle-sheath interface (Botha, 1992). The
necessity for metabolite transport between the mesophyll
and bundle sheath also sets limits on the amount of
mesophyll tissue that can be functionally associated with
bundle sheath tissue. Close contact between the two is
required, dictating the proximity of mesophyll and bundle
sheath cells and, therefore, influencing leaf structure.
For this reason, the leaf thickness is limited in C4 plants
and the interveinal distance (i.e. the number of mesophyll
cells between adjacent bundle sheaths) is usually smaller
than in the leaves of C3 plants (Hattersley, 1992). Thus
C3 plants engineered to express C4 photosynthesis between
the mesophyll and bundle sheath would be compromised
with regard to metabolite transport if the interveinal
distance were large.
The intracellular exchange of metabolites occurs at a
much greater rate than in C3 plants and C4 plants possess
translocators in the chloroplasts and mitochondria with
unique, or considerably altered, kinetic properties that
can transport key metabolites of the C4 cycle, such as
the C4 acids, malate and aspartate, as well as pyruvate,
oxaloacetate and PEP (Leegood, 2000). For example, the
exchange of inorganic phosphate, PEP, glycerate-3-P, and
triose-P occurs in chloroplasts of C3 and C4 plants via the
phosphate translocator. The affinity for PEP of this
translocator is 55-fold higher than that of the phosphate
translocator in C3 chloroplasts (Gross et al., 1990).
Again, in C3 and C4 plants, the dicarboxylates (oxaloacetate, malate, 2-oxoglutarate, glutamate, and aspartate)
undergo counter-exchange across the chloroplast envelope on the dicarboxylate translocator (Day and Hatch,
1981). However, in malate-forming C4 plants, such as
maize, oxaloacetate uptake would not occur when
oxaloacetate concentrations are several orders of magnitude less than malate concentrations. There is, therefore,
an additional high affinity oxaloacetate carrier in maize
mesophyll chloroplasts which is little affected by malate.
Although this carrier is also present in chloroplasts of
the C3 plant, spinach, it has a very much lower capacity
(Hatch et al., 1984). Clearly, limitations imposed by
C3 transporters on transport of intermediates of the C4
cycle between the cytosol and the chloroplast could
easily arise. Thus it would seem desirable to introduce
specific transporters in any programme to engineer a
CO2-concentrating mechanism.
Specialization of the functions of the mesophyll
and bundle-sheath has required modification of their
processes. For example, specialization of electron transport pathways in the bundle-sheath chloroplasts of
NADP-malic enzyme plants has led to a predominance
of photosystem 1-dependent cyclic electron transport
leading to ATP formation (Chapman et al., 1980), and
a low photosystem 2 activity. This means that NADPH
is only generated by NADP-malic enzyme, which is sufficient to reduce only 50% of the glycerate-3-P generated
by Rubisco in the bundle sheath. All C4 sub-types,
therefore, possess the enzymes for glycerate-3-P reduction
in the mesophyll chloroplasts (Hatch and Osmond, 1976).
However, even in those C4 sub-types that have photosystem 2 in the bundle-sheath, glycerate-3-P is reduced in
the mesophyll. It can thus be inferred that the glycerate3-Putriose-P shuttle between the bundle-sheath and mesophyll serves an important function in C4 metabolism. This
could be (i) a means of ensuring Hq transport, and
hence charge balance, between the two cell types; (ii) to
decrease the amount of reductant (NADPH) required
C4 photosynthesis
for reduction of glycerate-3-P in the bundle-sheath and
hence a decrease in photosynthetic O2 evolution in
the bundle sheath, thus improving the CO2uO2 ratio,
favouring Rubisco carboxylation over oxygenation;
(iii) co-ordination of the Benson–Calvin cycle and C4
cycle turnover. Co-ordination of activities in the mesophyll and bundle-sheath requires metabolic cross-talk
to occur. For example, triose phosphates and hexose
phosphates signal between the C3 and C4 cycles by
acting as positive effectors (‘metabolite messages’) of
PEP carboxylase activity (Chollet et al., 1996; Walker
and Leegood, 1999).
Just as mitochondria in leaves of C3 plants have
developed a high capacity to decarboxylate the glycine
generated by photorespiration (Douce et al., 2001),
mitochondria have a specialized role in photosynthesis
in both NAD-malic enzyme and PEPCK-type C4 plants,
which use NAD-malic enzyme to decarboxylate malate
in the bundle sheath mitochondria (Carnal et al., 1993).
Since photosynthetic fluxes vastly exceed respiratory
fluxes, this requires some uncoupling of C4 acid decarboxylation from normal mitochondrial metabolism
by engagement of the alternative, cyanide-insensitive
pathway of respiration (Agostino et al., 1996).
Aquatic C4 photosynthesis
The availability of inorganic carbon for photosynthesis in
water is limited by diffusion (104 lower than in air) and
pH. In addition, ambient O2 concentrations may rise to
twice those in air-saturated water. These are conditions
which potentially lead to high Rubisco oxygenase activity
and hence high rates of photorespiration. As a result,
many aquatic photosynthesizers have developed a variety
of CO2-concentrating mechanisms. In the majority of
cases, in algae, cyanobacteria and many aquatic angiosperms, these involve biophysical solutions (the uptake
of CO2 or bicarbonate or proton extrusion to convert
bicarbonate to CO2). However, at least three members of
the Hydrocharitaceae, Hydrilla verticillata, Egeria densa
(Casati et al., 2000) and Elodea canadensis (de Groote and
Kennedy, 1977) show evidence of C4 metabolism. Elodea
species and Egeria densa additionally have a mechanism
of apoplastic acidification that enhances the conversion
of HCO3 to CO2 (van Ginkel et al., 2001). There is also
evidence for C4 metabolism in a marine macroalga,
Udotea flabellum (Reiskind and Bowes, 1991) and in a
marine diatom, Thalassiosira weissflogii (Reinfelder et al.,
2000; though see Johnston et al., 2001). The best studied
of these is Hydrilla verticillata, which is a freshwater,
submerged angiosperm, a common introduction to freshwater lakes in the USA. In winter, H. verticillata plants
are scattered in open water, but in summer they form
dense mats of vegetation just below the surface. Plants in
585
these mats encounter low dissolved inorganic carbon,
especially low CO2, alkaline pH values, high temperatures, elevated O2 concentrations, and high irradiance
(Reiskind et al., 1997). When growing under these conditions, C4-like characteristics are induced, accompanied
by low CO2 compensation points, low rates of photorespiratory CO2 release, minimal inhibition of photosynthesis by O2, and increased net photosynthesis
(Salvucci and Bowes, 1981). This is accompanied by a
suite of metabolic changes characteristic of C4 photosynthesis, including the induction of PEP carboxylase
(Salvucci and Bowes, 1981), pyruvate, Pi dikinase,
NADP-malic enzyme, and aminotransferases (Magnin
et al., 1997), and transfer of label from C4 acids into
intermediates of the Benson–Calvin cycle (Salvucci and
Bowes, 1983). PEP carboxylase is cytosolic, whereas
PPDK, NADP-malic enzyme and NADP-malate
dehydrogenase are predominantly chloroplastic (Magnin
et al., 1997) (Fig. 3). However, the structural features of
H. verticillata show that it lacks the Kranz anatomy
characteristic of terrestrial C4 species (Reiskind et al.,
1997). The lamina of the H. verticillata leaf is only two
cells thick and no compartmentation of PEP carboxylase
and Rubisco into separate cells has been detected. Direct
measurements of the internal carbon pool indicated about
a 5-fold concentration over that in the surrounding water
(equivalent to about 400 mM CO2 in the chloroplast),
but no such concentration in uninduced ‘C3’ plants
(Reiskind et al., 1997). This rules out the notion that,
Fig. 3. The C4-like CO2-concentrating mechanism in the aquatic plant,
Hydrilla verticillata. PEP carboxylase (PEPC) in the cytosol fixes
bicarbonate, presumably generated from CO2 by carbonic anhydrase
(CA), and produces malate, which is decarboxylated in the chloroplast
by NADP-malic enzyme (NADP-ME) and the released CO2 is fixed by
Rubisco in the Benson–Calvin cycle. PEP is regenerated from pyruvate
by pyruvate, Pi dikinase (PPDK).
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Leegood
rather than having an active CO2-concentrating mechanism, H. verticillata is simply refixing photorespired CO2, as
in some C3–C4 intermediates, such as Moricandia arvensis
(Monson and Rawsthorne, 2000).
There is the evidence that the C4 mechanism is also
inducible in the amphibious leafless sedge, Eleocharis
vivipara, which has C3 biochemical traits under submerged conditions, but develops C4 biochemical traits,
as well as Kranz anatomy, under aerial conditions, a
process regulated by abscisic acid (Ueno, 1998). A C4
system lacking Kranz anatomy also appears to operate in
aquatic leaves of Orcuttia spp., which is a grass that
germinates and establishes in seasonal pools, followed
by a short aerial phase of growth after pools dry out
(Keeley, 1998).
Could a single-celled C4 system work in air?
A number of attempts are being made to introduce the
enzymic components of C4 photosynthesis into C3
plants, such as rice, tobacco and potato. These are based
on the H. verticillata system (i.e. PEP carboxylation in
the mesophyll and decarboxylation of C4 acids in the
chloroplast) (Fig. 3) and the premise that a single-celled
CO2-concentrating mechanism could operate in the
mesophyll cells of a C3 leaf in air.
Single cell CO2-concentrating mechanisms are effective
in algae and cyanobacteria. However, it must be emphasized that both have internal compartments within
the chloroplast (the pyrenoid in the algae and some
bryophytes and the carboxysome in cyanobacteria) with a
low CO2 permeability (Table 1) that prevent CO2 leakage
from the site of CO2 release (Badger and Spalding,
2000; Smith and Griffiths, 2000) and both function in
the aquatic environment. How does a single-cell C4-like
system operate to concentrate CO2 in the vicinity of
Rubisco in H. verticillata? There is, as yet, no evidence
for reduced membrane permeability or for any internal
structures that could concentrate CO2 within the chloroplast of H. verticillata. Although biological cells and
membranes show considerable variability in their permeability to CO2 (Table 1), perhaps partly influenced by
the presence of aquaporins (Nakhoul et al., 1998; Prasad
et al., 1998), at present it seems likely that the major
factor tending to prevent CO2 loss from the cells is the
diffusive resistance provided by the unstirred layer at the
boundary between the leaf and the environment (Keeley,
1998). This situation would also obtain in Orcuttia in
its submerged state but, additionally, in Orcuttia the
centripetal arrangement of mesophyll chloroplasts means
that chloroplasts are placed at the greatest possible
distance from the environment, which may facilitate the
operation of this single-celled C4 system both in water
and in air (Keeley, 1998).
It has only recently emerged that a fully terrestrial
plant, the hygrohalophytic chenopod Borszczowia
aralocaspica, has a single-cell CO2-concentrating mechanism. One means of identifying the presence of a C4-like
CO2 concentrating mechanism is to measure carbon
isotope composition. Differences in carbon isotope
composition arise because Rubisco discriminates against
naturally occurring 13CO2 much more than PEP carboxylase. Hence C3 plants are depleted in 13C, with carbon
isotope signatures ranging from about 20 to 35ø,
compared with C4 plants with carbon isotope signatures
ranging from about 10 to 14ø (Cerling, 1999). The
d13C values of B. aralocaspica were 13.03ø from young
stems, and 13.78ø from leaves (Freitag and Stichler,
2000), consistent with the occurrence of C4 photosynthesis. In addition, photosynthesis in B. aralocaspica
was not inhibited by O2, indicating the absence of photorespiration (Voznesenskaya et al., 2001). B. aralocaspica
has a novel type of leaf anatomy with single-layered
chlorenchyma in which elongated cells surrounding the
central water storage tissue are radially arranged (Freitag
and Stichler, 2000). At the inner, centripetal, end there are
large chloroplasts that are granal, have starch and high
amounts of Rubisco, and there are no intercellular air
spaces. However, the centrifugal end is exposed to the
intercellular space. There are some chloroplasts along
the radial walls of the cells towards the centrifugal end,
but these have few grana, little starch and low Rubisco.
Immunolocalization has shown that NAD-malic enzyme
is specifically located in the mitochondria at the centripetal
end of the cell, while PEP carboxylase is distributed
throughout the cytosol (Voznesenskaya et al., 2001). The
single-layered photosynthetic tissue is thus dimorphic and
combines the essential anatomical and functional characteristics of a two-layered chlorenchyma of regular C4
plants. It is inferred that the ‘bundle-sheath’ reactions
occur at the inner end of the cell, and that the ‘mesophyll’
reactions occur at the outer, centrifugal end. Clearly, there
are a lot more questions to answer as to mechanism, but
presumably it is able to function as a CO2-concentrating
mechanism in air because of the long diffusion path from
one end of the cell to the other. So the answer to the
question ‘Could a single-celled C4 system work in air?’ is
apparently ‘yes’, but only with leaf anatomy substantially
different from that of a conventional C3 leaf.
Biotechnological approaches to
C4 photosynthesis
The simplest theoretical single-celled system that could be
engineered into a C3 leaf would be PEP carboxylation in
the cytosol, oxaloacetate transport into the chloroplast,
decarboxylation of oxaloacetate to PEP by PEP carboxykinase in the chloroplast, followed by transport of
C4 photosynthesis
PEP back to the cytosol. This could be achieved at the
expense of only 1ATP per CO2 transferred, although no
C4 plant is known to possess such a system. Alternatively,
the Hydrilla system utilizes two more steps with the
decarboxylation of malate by NADP-malic enzyme and
the regeneration of PEP from pyruvate by pyruvate,
Pi dikinase (Fig. 3). This would involve expressing
carbonic anhydrase in the cytosol to ensure a supply of
bicarbonate to PEP carboxylase, PEP carboxylase in
the cytosol, NADP-malic enzyme and pyruvate, Pi
dikinase in the chloroplast, and transporters for PEP
and oxaloacetate to ensure that the operation of the
cycle was not limited by intracellular transport. It might
be that the activity of NADP-malate dehydrogenase
would also be insufficient. Attempts have been made to
introduce these various steps, mainly singly, into the
leaves of C3 plants. In all these attempts, it must be borne
in mind that three of these enzymes (PEP carboxylase,
PEP carboxykinase and pyruvate, Pi dikinase) are regulated by phosphorylation, so that the introduced enzyme
may be down-regulated in vivo by post-translational
regulation, thus defeating attempts at over-expression.
This can be avoided by introducing unregulated forms of
the enzymes, either by site-directed mutagenesis or from
other organisms.
Introducing a single enzyme, or even an incomplete
portion of the C4-cycle, is, of course, unlikely in itself to
have a large impact on photosynthesis. However, there is
some evidence that manipulations have led to the desired
redirection of fluxes. Maize PEP carboxylase was overexpressed in tobacco plants leading to 2-fold higher
activities. Transgenic tobacco plants had significantly
elevated levels of titratable acidity and malic acid, consistent with this increased expression, but no physiological
changes with respect to photosynthetic rate or CO2
compensation point (Hudspeth et al., 1992). The authors
concluded that PEP carboxylase was probably working
maximally at only a few per cent of the rate of photosynthesis. Ku et al. introduced the intact gene of maize
PEP carboxylase into rice giving remarkably high levels
of expression (Ku et al., 1999). The activities of PEP
carboxylase in leaves of some of these transgenic rice
plants were 2–3-fold higher even than those in maize,
and the enzyme accounted for up to 12% of the total
leaf-soluble protein. It is clear from this work that
increasing the amounts of PEP carboxylase in isolation
does not have dramatic effects on photosynthesis,
although it may alter stomatal conductance (Ku et al.,
2000). The transgenic rice plants exhibited reduced
O2 inhibition of photosynthesis, but this is probably
due to effects of phosphate recycling than effects on
photorespiration (Matsuoka et al., 2000; Leegood and
Furbank, 1986). It is likely that the PEP carboxylase
introduced into rice is largely inactive in vivo because
of down-regulation by dephosphorylation (Chollet et al.,
587
1996). Interestingly, in their early work with Atriplex
crosses, Björkman et al. already concluded that elevated
PEP carboxylase activity by itself (up to a 10-fold
increase) had no effect on the CO2 compensation point,
the photosynthetic rate, or the inhibitory effects of O2
on photosynthesis (Björkman et al., 1971).
Attempts to engineer the Hydrilla-type system into
potato suggest that modest expression of PEP carboxylase
and NADP-malic enzyme, either singly or in combination, can influence photorespiratory characteristics. For
example, substantial over-expression of PEP carboxylase
from Corynebacterium glutamicum in potato led to an
increase in the rate of dark respiration and a small
decrease in C*, the CO2-compensation point measured in
the absence of dark respiration (Häusler et al., 1999).
Double transformants with an additional 3–5-fold overexpression of Flaveria pringlei NADP-malic enzyme in the
chloroplast showed a temperature-dependent decrease
in the electron requirement for CO2 assimilation, again
suggesting a slight suppression of photorespiration (Lipka
et al., 1999). Double transformants of potato (PEP
carboxylase and NADP-malic enzyme) exhibited the
most consistent attenuating effect on photorespiration,
as shown by reductions in C* as well as temperature and
oxygen effects on photosynthesis. However, these are
most likely a result of local changes in CO2 concentration
induced by increasing respiration (Häusler et al., 2002).
Suzuki et al. over-expressed an unregulated phosphoenolpyruvate carboxykinase (PCK) from the C4 plant,
Urochloa panicoides, in the chloroplasts of rice plants
(Suzuki et al., 2000). In 14CO2 labelling experiments, up
to 20% of the radioactivity was incorporated into C4
acids (malate, oxaloacetate, and aspartate) in leaves of
transgenic plants, as compared with about 1% in excised
leaves of control plants. When 14C-malate was fed to
excised leaves the extent of incorporation of radioactivity
into sucrose was 3-fold greater in transgenic plants
than in control plants and the level of radiolabelled
aspartate was significantly lower in transgenic plants.
Thus, expression of PCK in rice chloroplasts led to a
partial change in carbon flow in mesophyll cells into a
C4-like photosynthetic pathway. In tobacco, introduction
of the PEP carboxykinase gene from the bacterium
Sinorhizobium meliloti, either singly or in combination
with PEP carboxylase, had little effect on photosynthetic
parameters (Häusler et al., 2001).
Potato plants over-expressing maize pyruvate, Pi
dikinase showed a decrease in pyruvate and an increase
in malate and a small, but significant decrease in
d13C, suggesting increased PEP carboxylation (Ishimaru
et al., 1998). There was, however, no change in CO2compensation point. In rice, over-expression of maize
pyruvate, Pi dikinase (Fukayama et al., 2001) has been
claimed to lead to a higher photosynthetic rate, associated
with higher stomatal conductance (Ku et al., 2000).
588
Leegood
Potentially useful pleiotropic effects have also occurred
in these manipulations. A 20–70-fold increase in maize
NADP-malic enzyme in rice leaves (located mainly in the
chloroplasts) led to aberrant chloroplast structure with
agranal thylakoid membranes, and an inverse correlation
between NADP-malic enzyme activity and chlorophyll
and photosystem II activity (Takeuchi et al., 2000). This
is particularly interesting in relation to the presence of
agranal chloroplasts in the bundle-sheath of NADP-malic
species. Hoewever, other studies of rice over-expressing
maize NADP-malic enzyme have also indicated a reduction in chlorophyll content, reduced growth and enhanced
photoinhibition (Tsuchida et al., 2001), probably resulting from over-reduction of the NADP pool as a result
of a high activity of the over-expressed enzyme in vivo
(Takeuchi et al., 2000; Tsuchida et al., 2001).
Conclusions
Leaving aside all the arguments about whether or not it is
desirable to engineer C4 photosynthesis into C3 plants
(Sheehy et al., 2000), it seems unlikely that attempts
to introduce single cell CO2-concentrating mechanisms
will be successful without also introducing some of the
structural characteristics of C4 photosynthesis, that is,
a compartment in which CO2 could be concentrated. If
the anatomical characteristics of C4 plants were to be
incorporated, this means understanding the complex
factors that regulate the development of the different
cell types in C4 plants and, although this may only be
controlled by a few genes, these have not yet been identified. Even after making the bundle-sheath of an existing
C3 plant green by enhancing chloroplast number, and
expressing the appropriate enzymes in the mesophyll
and bundle-sheath, the leaf structure would still be
inappropriate for the operation of C4 photosynthesis
because of poor cell-to-cell communication. Equally,
engineering a single-celled C4 system, such as that found
in Borszczowia, would require a reworking of C3 leaf
structure. The alternative is to take a different approach
to reducing photorespiration. One approach would be
to introduce pyrenoids or carboxysomes (intracellular
compartments for the concentration of CO2 that are
found in algae and cyanobacteria) into the chloroplasts
of C3 plants. Another would be to move the location
of photorespiratory glycine metabolism to the bundlesheath, taking as a working example the suppression
of photorespiration that occurs in C3–C4 intermediates
(Winzer et al., 2001; Monson and Rawsthorne, 2000). The
last alternative would be to express, in C3 plants,
improved forms of Rubisco, notably those from rhodophyte algae in which the relative specificity for CO2
compared to O2 is higher than that of higher plant
Rubisco (Whitney et al., 2001).
Acknowledgements
Research in Sheffield into C4 photosynthesis and photorespiratory metabolism was supported by the Biotechnology
and Biological Sciences Research Council, UK.
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