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Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:297–314
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MOLECULAR ENGINEERING
OF C4 PHOTOSYNTHESIS
Makoto Matsuoka
BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan;
e-mail: [email protected]
Robert T Furbank
CSIRO Plant Industry, G.P.O. Box 1600, Canberra ACT 2601, Australia;
e-mail: [email protected]
Hiroshi Fukayama and Mitsue Miyao
Laboratory of Photosynthesis, National Institute of Agrobiological Resources, Kannondai,
Tsukuba 305-8602, Japan; e-mail: [email protected]
Key Words C3 plants, C4 plants, carbon metabolism, evolution, transgenic plants
■ Abstract The majority of terrestrial plants, including many important crops such
as rice, wheat, soybean, and potato, are classified as C3 plants that assimilate atmospheric CO2 directly through the C3 photosynthetic pathway. C4 plants such as maize
and sugarcane evolved from C3 plants, acquiring the C4 photosynthetic pathway to
achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. The recent application of recombinant DNA technology has made considerable
progress in the molecular engineering of C4 photosynthesis over the past several years.
It has deepened our understanding of the mechanism of C4 photosynthesis and provided valuable information as to the evolution of the C4 photosynthetic genes. It also
has enabled us to express enzymes involved in the C4 pathway at high levels and
in desired locations in the leaves of C3 plants for engineering of primary carbon
metabolism.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
METABOLIC ENGINEERING OF C4 ENZYMES IN C4 PLANTS . . . . . . . . . . . .
Rate-Limiting Reactions in C4 Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . .
Transgenic C4 Plants and the CO2 Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MOLECULAR ENGINEERING OF C4 ENZYMES IN C3 PLANTS . . . . . . . . . . .
How to Overproduce C4 Enzymes in the Leaves of C3 Plants . . . . . . . . . . . . . . . .
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Physiological Impacts of Overproduction of C4 Enzymes in C3 Plants . . . . . . . . . 307
FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
INTRODUCTION
The majority of terrestrial plants, including many important crops such as rice,
wheat, barley, soybean, and potato, assimilate atmospheric CO2 directly through
the C3 photosynthetic pathway, also known as the Calvin cycle, and these are classified as C3 plants. The enzyme of primary CO2 fixation in this pathway, ribulose
1,5-bisphosphate carboxylase/oxygenase (Rubisco), reacts not only with CO2 but
also with O2, leading to photorespiration, which essentially wastes assimilated
carbon. Under current atmospheric conditions, potential photosynthesis in C3
plants is suppressed by oxygen by as much as 40%. The extent of suppression
further increases under stress conditions such as drought, high light, and high temperature, through a decline of the CO2 concentration inside leaves due to closure
of stomata. C4 plants such as maize, sorghum, and sugarcane have evolved a novel
biochemical mechanism to overcome photorespiration. In addition to the C3 pathway, they use the C4 photosynthetic cycle to elevate the CO2 concentration at the
site of Rubisco and thus suppress its oxygenase activity. This mechanism enables
C4 plants to achieve elevated photosynthetic capacity particularly at higher temperatures, of up to twice as high as that of C3 plants, in addition to higher waterand nitrogen-use efficiencies (for reviews of C4 photosynthesis, see 17, 22, 40a).
Consequently, it has long been postulated that the transfer of C4 traits to C3 plants
could improve the photosynthetic performance of C3 species.
Leaves of C4 plants have two types of photosynthetic cells, the mesophyll cell
(MC) and bundle sheath cell (BSC). While all the photosynthetic enzymes are
confined in MCs in C3 plants, they are localized in MCs and/or BSCs in C4 plants
(Table 1). In addition, C4 plants show extensive venation, with a ring of BSCs
surrounding each vein and an outer ring of MCs surrounding the bundle sheath.
This unique leaf structure, known as Kranz anatomy, and the cell-specific compartmentalization of enzymes are essential for operation of the C4 pathway.
The initial fixation of CO2 in the C4 pathway occurs in the MC cytosol by phosphoenolpyruvate carboxylase (PEPC) to form the C4 acid oxaloacetate (OAA).
OAA is either reduced to malate by NADP-malate dehydrogenase (NADP-MDH)
or transaminated to aspartate by aspartate aminotransferase (AspAT). The resultant
C4 acid is transported to the BSCs and then decarboxylated to release CO2 in the
vicinity of Rubisco. The decarboxylation reaction is catalyzed by one or more of
the three enzymes, namely, NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK), and C4
plants are classified into three subtypes depending on the major decarboxylation
enzyme. A C3 acid generated by the decarboxylation is shuttled back to MCs
to regenerate the primary CO2 acceptor phosphoenolpyruvate (PEP) by pyruvate,orthophosphate dikinase (PPDK) in the MC chloroplasts.
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TABLE 1 Location of major photosynthetic enzymes in leaves of C4 plants
Enzyme
Gene
C4 subtype(s)
Location
Function
Rubisco (C3)
rbc
All
BSC, Chlt
Net CO2 fixation
PEPC (C4)
Ppc
All
MC, Cyt
Initial CO2 (HCO3−) fixation
PPDK (C4)
Pdk
All
MC, Chlt
Regeneration of PEP
NADP-MDH (C4)
mdh
NADP-ME
MC, Chlt
OAA → malate
NADP-ME (C4)
Me
NADP-ME
BSC, Chlt
Decarboxylation of malate
NAD-ME (C4)
NAD-ME
(PEP-CK)
BSC, Mit
Decarboxylation of malate
AspAT (C4)
NAD-ME
PEP-CK
MC, Cyt;
BSC, Mit
OAA → asparate (MC)
Asparate → OAA (BSC)
PEP-CK (C4)
PEP-CK
(NADP-ME)
BSC, Cyt
Decarboxylation of OAA
CA (C4)
All
MC, Cyt
CO2 → HCO3−
Enzymes mentioned in this article are listed. MC, mesophyll cells; BSC, bundle sheath cells; Cyt, cytosol; Chlt,
chloroplasts; Mit, mitochondria.
The enzymes involved in the C4 pathway (C4 enzymes) had previously been
considered to be specific for C4 plants, since the activities of homologues are low
in C3 plants and their kinetic properties are usually different from those of C4
enzymes. However, recent comparative studies have revealed that C3 plants have
at least two different types of genes, one encoding enzymes of “housekeeping”
function and the other very similar to the C4 photosynthetic genes in C4 plants,
although expression of the latter is very low or even undetectable in C3 plants.
Based on this finding, it is postulated that the C4-specific genes evolved from a set
of preexisting counterpart genes in ancestral C3 plants, with some modifications
in expression patterns in leaves and kinetic properties of enzymes (33, 40).
The recent application of recombinant DNA technology to plant metabolism
has considerably advanced our understanding of the regulation of photosynthesis (18). By altering the levels and properties of key enzymes in photosynthesis in transgenic C4 plants, we are now able to deepen our understanding of
the mechanism of C4 photosynthesis. Cell-specific expression of C4 photosynthetic genes can also be probed using reporter gene constructs in transgenic plants
(9, 33). Our understanding of the evolution of photosynthetic carbon metabolism
can be advanced by examining the consequences of the transfer of C4-specific
genes to transgenic C3 plants. Lastly, a great challenge is to use these techniques and C4 genes to alter carbon metabolism of C3 crop plants to answer
fundamental questions concerning agronomic performance and to improve crop
productivity. Here we review progress in these key areas of C4 photosynthesis
research.
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METABOLIC ENGINEERING OF C4 ENZYMES
IN C4 PLANTS
The capacity to alter photosynthesis in transgenic plants with a high degree of
precision has provided a powerful tool for studying rate-limiting processes in photosynthesis and carbon partitioning in vivo (see 18). This technology, using the
C4 dicot Flaveria bidentis (8) along with the isolation of C4 mutants of Amaranthus edulis requiring high CO2 for growth (see 10), has allowed the analysis
of control points in the C4 pathway in intact plants. In this section we review
the contribution of these approaches to our understanding of C4 metabolism and
mechanism.
Rate-Limiting Reactions in C4 Photosynthesis
One of the great challenges in plant biochemistry has been to transfer the vast
amount of knowledge on enzyme kinetics in vitro to the control of flux through
metabolic pathways in vivo. Using antisense, cosuppression, ectopic overexpression, and the mutational approach described above, we now have a relatively clear
picture of the role individual enzymes play in controlling photosynthetic flux in C4
plants. Table 2 summarizes this work by presenting control coefficients (Cj) for the
individual enzymes examined so far. These coefficients were mostly determined
at saturating light and ambient CO2. The significance of control coefficients has
been reviewed elsewhere (1) but a Cj of one indicates that flux is fully controlled
at this step in the pathway, whereas a value of zero indicates no control over flux.
The results summarized in Table 2 indicate that the bulk of control in C4 photosynthesis lies with the three enzymes Rubisco, PPDK, and PEPC. Experiments
where expression of the gene encoding the small subunit of Rubisco (rbcS) was
reduced by antisense in Flaveria indicate that at high light, a control coefficient of
0.5 to 0.6 is likely for this enzyme (15, 16). This result was somewhat surprising
as it has been suggested that the high CO2 concentration in BSCs has allowed C4
plants to reduce the amount of Rubisco in BSCs, relative to that in C3 plants (one
TABLE 2 Control coefficients (Cj) for photosynthetic enzymes in C4 plants determined
by measuring CO2 assimilation under saturating illumination and ambient CO2
Enzyme
C4 species
Technique
Cj
References
Rubisco
F. bidentis
Antisense RNA
0.5–0.6
15, 16
PPDK
F. bidentis
Antisense RNA
0.2–0.4
15
PEPC
A. edulis
Mutation
0.35
2, 10
NAD-ME
A. edulis
Mutation
∼0
11
NADP-MDH
F. bidentis
Cosuppression
∼0
47
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third to one quarter of C3 levels), without affecting photosynthesis, resulting in
greater nitrogen-use efficiency (41). It appears that this evolutionary optimization
of Rubisco level has poised nitrogen investment in this protein to the minimum
effective level in wild-type plants. As with C3 plants, it would be instructive to
elevate Rubisco levels in transgenic C4 plants; however, the overexpression of the
chloroplast-encoded large subunit of Rubisco currently provides a technical barrier
to these experiments.
Control of photosynthesis by PPDK and PEPC is more difficult to assess. Both
these enzymes are subject to complex regulation by reversible protein phosphorylation and in the case of PEPC, mutants have been shown to increase the enzyme
activation state in response to decreased enzyme levels (10). In the case of PEPC,
a control coefficient of 0.35 was calculated for Amaranthus mutants at saturating
light and ambient CO2 (2). A similar value was found for PPDK in transgenic
Flaveria containing an antisense gene construct targeted to this enzyme (15). The
fact that the Cj values for these three enzymes sum to greater than one may be
due to the different growth conditions used in each case or possibly interspecific
or decarboxylation-type variations in enzyme levels.
Two of the enzymes in C4 photosynthesis examined thus far appear to exert
little or no control over photosynthetic flux in vivo: NADP-MDH (47) and NADME (11). In the case of NADP-MDH, transgenic Flaveria containing antisense or
cosuppression constructs showed no effect of reduced enzyme level until plants
contained less than 10% of wild-type levels of the protein (47). Even after it is
taken into account that these plants showed increased enzyme activation state to
compensate for reduced enzyme levels, Cj for this thioredoxin-regulated enzyme
was effectively zero under all conditions examined (47). Similarly, in heterozygous
Amaranthus mutants with an approximately 50% reduction in NAD-ME activity,
no effect on photosynthesis could be detected, indicating that this enzyme effectively exerts no control over photosynthetic flux (11).
Although the experiments described above in which high control coefficients are
observed indicate where control of flux in C4 photosynthesis lies, the observations
that several “key enzymes” appear to have no regulatory role are equally intriguing.
NADP-MDH is highly regulated by light through the thioredoxin-mediated reduction of multiple disulfide bridges (see 17). NAD-ME activity is also regulated, in
this case allosterically, by a number of metabolites including adenylates (14). Yet
modulation of these enzymes, apart from a crude on/off mechanism, could have
no effect on photosynthetic flux in vivo. These observations are important in that
they should engender a degree of caution when espousing the importance of a key
enzyme in regulation of any metabolic pathway and raise the question as to why
sophisticated mechanisms of enzyme regulation are necessary for such enzymes.
One possible explanation lies in the evolutionary origins of C4 photosynthesis.
There is not a single enzyme in the C4 pathway that does not have a homologue
in C3 plants, leading to the hypothesis that the genes encoding these proteins have
been recruited for the C4 process [see Introduction; (9, 33)]. The presence in C4
plants of these housekeeping enzymes at levels up to 100-fold higher than those
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found in C3 leaf cells, often still in their original C3 compartment, could play havoc
with all manner of pathways of secondary metabolism (see 15). It is tempting to
postulate that regulation of these nonlimiting enzymes preserves flux through minor pathways by conserving metabolites from the massive flood of carbon through
C4 photosynthesis.
Transgenic C4 Plants and the CO2 Pump
A long-standing focus of interest in C4 photosynthesis has been the CO2 concentrating mechanism and the barrier to CO2 diffusion afforded by the bundle
sheath/mesophyll interface (see 49). This aspect of the C4 mechanism is particularly pertinent to the introduction of C4 traits into C3 plants that lack the Kranz
leaf anatomy common to terrestrial C4 plants. It has long been postulated that the
relative levels of the enzymes of the CO2 pump in MCs and the enzymes of the
Calvin cycle in BSCs, along with the permeability characteristics of BSCs, largely
determine the efficiency with which C4 photosynthesis operates (49). Transgenic
plants with altered PEPC/Rubisco ratios (45, 50) or altered bundle sheath inorganic
carbon composition (35) provide the opportunity to test these hypotheses.
If the content of Rubisco in a C4 leaf is progressively reduced without a commensurate effect on PEPC levels (as is the case in the transgenic Flaveria discussed
above), one would intuitively expect the bundle sheath CO2 concentration to rise.
This would result in an increase in leakage of CO2 from the bundle sheath and
a decline in photosynthetic efficiency per net CO2 fixed. This is, in fact, what
is observed when the Rubisco antisense Flaveria transgenics are analyzed using
carbon isotope discrimination (50) or chlorophyll fluorescence and gas exchange
(45). Carbon isotope discrimination allows the calculation of a leakiness parameter known as φ, which is the fraction of CO2 generated in C4 acid decarboxylation
that subsequently leaks out of the bundle sheath (see 49). In transgenic Flaveria
with a 40% reduction in Rubisco, this leakiness parameter increased by 50%. The
increased ATP required to support this CO2 leakage was also evident in the quantum efficiency of the photosystems where plants with a 70% reduction in Rubisco
content required approximately 28 quanta per CO2 fixed compared with 17 for
wild-type leaves at saturating light in air (45).
It has been postulated that an important requirement of the CO2 concentrating
mechanism of C4 photosynthesis is the lack of equilibration of CO2 and bicarbonate
in BSCs (29). This hypothesis is supported by the lack of appreciable carbonic
anhydrase (CA) activity in BSCs of any C4 plant (6). Theoretically, by reducing the
bicarbonate content of the bundle sheath inorganic carbon pool, the concentration
of CO2 (the active species for fixation by Rubisco) is optimized and leakage of
bicarbonate to MCs through plasmodesmata minimized (29). This hypothesis has
been tested and the energetic ramifications assessed by ectopically expressing CA
from tobacco in BSCs of Flaveria (35). Transgenic plants with two- to fivefold
increases in CA activity in BSCs showed similar increases in leakiness to the
Rubisco antisense transgenic plants discussed above and an approximately 20%
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decrease in light-saturated photosynthesis (35). These experiments indicate the
importance of seemingly minor aspects of biochemical specialization of the C4
pathway in determining the efficiency of the process.
MOLECULAR ENGINEERING OF C4 ENZYMES
IN C3 PLANTS
How to Overproduce C4 Enzymes in the Leaves of C3 Plants
Previously, attempts have been made to transfer C4 traits to C3 plants by conventional hybridization between C3 and C4 plants (4). However, this approach was
available only in several plant genera such as Panicum, Moricandia, Brassica,
Atriplex, and Flaveria. Moreover, most C3-C4 hybrids showed infertility due to
abnormal chromosome pairing and/or genetic barriers. Recent developments in
plant genetic engineering have enabled us to introduce the desired genes encoding
C4 enzymes into C3 plants. In the past several years a variety of “C4 transgenic”
C3 plants have been produced (Table 3).
Enzymes Located in the Mesophyll Cells of C4 Plants The first attempt of this
kind used a chimeric gene construct containing a cDNA of the maize C4-specific
PEPC (Ppc cDNA) fused to the 50 - and 30 -flanking sequences of the chlorophyll a/b
binding protein gene (Cab) from Nicotiana plumbaginifolia (25). The introduction of this chimeric gene into tobacco increased the PEPC activity in the leaves to
2.2-fold that of nontransformants, but the levels of transcripts and protein in these
transformants were far below those in maize. Similarly, the expression of a cDNA
for the C4 enzyme under the control of strong promoters such as Cab, rbcS, and
Cauliflower mosaic virus 35S promoters led to only two- to fivefold increases in
the activity of PEPC (20, 31), PPDK (12, 27, 44), and NADP-MDH (19). In these
transformants, the level of the enzyme protein was low and only detectable by immunoblotting. The expression of bacterial Ppc genes from either Escherichia coli
or Corynebacterium glutamicum under the control of the 35S promoter increased
the enzyme activity of transgenic potato leaves but the extent of increase was less
than several fold, a value almost comparable to that obtained with the higher plant
Ppc cDNA under the control of the 35S promoter (20).
To raise the expression level of the C4 enzymes, sequences that have enhancerlike effects were included in the introduced gene (20, 28). Gehlen et al (20) examined the effects of the 50 -untranslated region (UTR) of the chalcone synthase gene
from parsley, and found that the expression of the Ppc gene from C. glutamicum
under the control of the 35S promoter was enhanced by its presence. The highest expression level, however, was still only fivefold that of nontransformants in
terms of PEPC activity. Thus, conventional strategies to express foreign genes in
transgenic plants did not dramatically increase the activity of C4 enzymes in the
leaves of C3 plants.
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TABLE 3 Increase in activities of C4 enzymes in the leaves of transgenic C3 plants
C4 enzyme
(Location in
C4 plants)
Host
C3 plant
PEPC (MC)
Tobacco
Potato
Rice
PPDK (MC)
Tobacco
Arabidopsis
Potato
Rice
Highest activitya
(Increase in fold)
References
Nicotiana Cab prom::maize
C4 cDNA::Nicotiana Cab
terminator
35S prom::maize C4 cDNA
35S prom::50 UTR::Corynebacterium gene
Intact maize C4 gene
2.2
25
2.4
5.4
31
20
110
32
35S prom::Mesembryanthemum FL CAM cDNA
Arabidopsis rbcS prom::maize
C4 cDNA
35S prom::maize C4 cDNA
Enhanced 35S prom::maize
C4 cDNA
Rice Cab prom::maize FL
C4 cDNA
Maize Pdk prom::maize FL
C4 cDNA
Intact maize C4 gene
1.6
44
2.4
27
4.0
5.4
27
28
5
13
5
13
40
13
Introduced construct
NADP-MDH
(MC)
Tobacco
35S prom::Sorghum FL
C4 cDNA
3
19
AspAT (MC)
Tobacco
35S prom::C4 Panicum
cDNA
Intact C4 Panicum gene
3.1
43
20
—b
Rice
AspAT (BSC)
Tobacco
35S prom::C4 Panicum
cDNA
3.5
43
NADP-ME
(BSC)
Potato
35S prom::C3 Flaveria
cDNA
Rice Cab prom::rice FL
C3 cDNA
Rice Cab prom::maize FL
C4 cDNA
7.1
34
5
48
30
48
70
46
0.5c
45a
0.5c
45a
Rice
PEP-CK
(BSC)
a
Maize Ppc prom::Urochloa
C4 cDNA
Maize Pdk prom::Urochloa
C4 cDNA
Highest enzyme activities among the primary transgenic plants are listed.
b
c
Rice
M Nomura & M Matsuoka, unpublished observation.
Highest activities of the secondary transgenic plants relative to the activity of Urochloa leaves are presented.
MC, mesophyll cells; BSC, bundle sheath cells; prom, promoter; FL, full-length.
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Another approach has been used to introduce the intact gene of C4 enzymes from
C4 plants into C3 plants. Our previous studies have demonstrated that the promoters
for maize C4-specific genes such as Ppc and Pdk can drive high-level expression
of a reporter gene in transgenic rice plants in an organ-specific, MC-specific, and
light-dependent manner as in maize (38–40). These results suggest that the rice
plant possesses the regulatory factors necessary for high-level expression of
C4-specific genes, and they imply that the introduction of the intact maize gene
would lead to high-level expression of C4 enzymes in rice leaves. As expected,
the introduction of the intact maize C4-specific Ppc gene containing all exons and
introns and its own promoter and terminator sequences led to high-level expression
of the PEPC protein in the leaves of transgenic rice plants (32). The majority (85%)
of the transgenic rice plants showed PEPC activity in the leaves 2- to 30-fold
that of nontransformants, whereas the remaining showed activity 30- to 110-fold
that of nontransformants, or 1- to 3-fold that of maize leaves. The level of the PEPC
protein accounted for 12% of total leaf soluble protein at most. In these transgenic
rice plants, the levels of transcripts and protein and the PEPC activity in the leaves
all correlated well with the copy number of the introduced gene. The levels of
transcripts per copy of the maize C4-specific Ppc gene were also comparable in both
maize and transgenic rice plants (32). These observations suggest that the maize
C4-specific Ppc gene behaves in a qualitatively and also quantitatively similar way
in both maize and transgenic rice plants. The introduction of the intact maize gene
was also effective in expressing another C4 enzyme, PPDK, in rice leaves. The
introduction of the intact maize C4-specific Pdk gene increased the PPDK activity
in rice leaves up to 40-fold that of nontransformants or about half of the activity
in maize (12). In a homozygous transgenic line, the PPDK protein accounted for
35% of total leaf soluble protein or 16% of total leaf nitrogen (12), much above
the levels of foreign protein in transgenic C3 plants reported previously.
Such high-level expression of the C4 enzymes in rice leaves could not be solely
ascribed to the transcriptional activity of the maize gene, since the expression of
a chimeric gene containing the full-length cDNA for the maize C4-specific PPDK
under the control of either the promoter of the maize C4-specific Pdk or the rice Cab
promoter increased the PPDK activity only up to several fold (12). It is suggested
that one or more of the introns or the terminator sequence of the maize gene, or a
combination of both, leads to high-level expression of the C4 enzyme by increasing
the stability of the transcript (12).
Maize and rice both belong to the Gramineae. Our recent study indicated that
the introduction of the intact gene from other C4 gramineous plants could also
lead to high-level expression of a C4 enzyme in transgenic rice plants. The activity
of the cytosolic form of AspAT in rice leaves was increased to reach 20-fold that
of nontransformants by introduction of the intact gene from Panicum miliaceum
(M Nomura & M Matsuoka, unpublished observations). P. miliaceum is classified
in the NAD-ME subtype of C4 plants, whereas maize is in the NADP-ME subtype.
Thus, the intact genes from C4 gramineous plants, irrespective of the C4 subtype,
will likely lead to high-level expression of the C4 enzymes in MCs of C3 gramineous
plants.
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This strategy, however, has some limitation in that transgenes from phylogenetically closely related plants have to be used to achieve high-level expression
of the C4 enzyme in C3 plants. The intact maize C4-specific Ppc gene was not expressed at high levels in tobacco leaves, because of incorrect transcription initiation
(25). Not only incorrect initiation and termination of transcription but also incorrect splicing could occur when genes from monocots are introduced into dicots
(see 21). Thus, phylogenetic distance may hamper the expression of genes from
C4 plants in the leaves of C3 plants.
Enzymes Located in the Bundle Sheath Cells of C4 Plants Unlike the C4 enzymes located in MCs of C4 plants, those located in BSCs can be expressed at
high levels in MCs of C3 plants by the introduction of a chimeric gene containing the full-length cDNA for the C4 enzyme fused to the Cab promoter, which
directs mesophyll-specific expression in C3 plants (42). The expression of the
maize C4-specific NADP-ME cDNA under the control of the rice Cab promoter
increased the activity of NADP-ME in rice leaves to 30- or 70-fold that of nontransformants (46, 48). The level of the NADP-ME protein was also increased to
several percent of total leaf soluble protein (46, 48). Such high-level expression
was unique to the cDNA for the C4-specific NADP-ME, and expression of the
cDNA for the C3-specific isoform increased the activity only several fold (34, 48).
The Flaveria C4-specific Me gene has a regulatory sequence in the 30 UTR that enhances its expression (37), and this sequence increases expression of a reporter gene
when combined with heterologous promoters in the leaves of both C4 Flaveria and
tobacco (S Ali & WC Taylor, unpublished information). The maize C4-specific Me
gene may therefore contain such an enhancer element for high-level expression
whereas the C3-specific gene does not. The expression of a cDNA of the C4-specific
PEP-CK of Urochloa panicoides under the control of the maize C4-specific Ppc
or Pdk promoter was also effective in increasing the activity of PEP-CK in MCs
of rice leaves (45a).
Recently, expression of the intact gene for C4 enzymes located in BSCs of C4
plants in C3 plants has also been addressed. When the intact gene for the mitochondrial AspAT of Panicum miliaceum, which is located in BSCs, was introduced into
rice, high AspAT activity was detected in vascular tissues and BSCs of transgenic
rice plants (M Nomura & M Matsuoka, unpublished observations). Similar results
were observed with the PEP-CK gene from Zoysia japonica and β-glucuronidase
activity under the control of the PEP-CK promoter, which was selectively detected
in vascular tissues and BSCs (M Nomura & M Matsuoka, unpublished information). These results demonstrate that the C4-specific genes for the BSC enzymes
can retain their property of cell-specific expression even in a C3 plant, rice, and they
therefore suggest that C3 plants have a regulatory mechanism for gene expression
of the BSC-specific C4 genes at their correct site. This fact is interesting in terms
of evolutionary aspects of C4 photosynthesis, but it also indicates that the strategy
to introduce intact C4-specific genes is not applicable to building the C4 pathway
solely in MCs of C3 plants.
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Physiological Impacts of Overproduction of C4 Enzymes
in C3 Plants
PEPC At present, there are four independent reports of transgenic C3 plants
that overproduce PEPC in the MC cytosol (Table 3): two independent reports
of transgenic tobacco plants expressing the maize C4-specific PEPC gene (25, 31),
transgenic potato expressing a bacterial PEPC gene from C. glutamicum (20),
and transgenic rice expressing the maize C4-specific PEPC gene (32). In the former
three cases, PEPC activities in the leaves of 2- to 5-fold greater than wild-type levels
were reported, whereas in the latter, activities up to 110-fold greater than wild-type
were observed. In general, PEPC from either higher plants or bacteria undergoes
regulation by various metabolite effectors, being inhibited by malate, aspartate,
and glutamate (see 17). Since concentrations of these inhibitors are high in the MC
cytoplasm of C3 plants, about 1 mM for malate and around 40 mM for aspartate and
glutamate (24), the PEPC activities of the transformants in vivo would be lower
than maximum extractable activities, especially when measured in the presence
of activators. Plant PEPC is also regulated by phosphorylation at a specific serine
residue near the N terminus that reduces sensitivity to these inhibitors (see 17).
The maize PEPC in transgenic rice leaves remained in its dephosphorylated and
less active form during illumination (13).
All the transformants analyzed to date show a higher level of malate (23, 25, 31)
or OAA (13) in the leaves compared with wild-types, an indication that the foreign PEPC is at least active in the leaves of these transformants. The level of
malate/OAA in these transformants, however, did not exceed twofold that of wildtype, irrespective of the expression level of PEPC in the C3 leaves. This is consistent
with the notion that PEPC is not fully active in these plants. It is also likely that
malate and OAA produced by the action of PEPC are metabolized inside the cell
or translocated from the leaves. The endogenous PEPC in MCs of C3 plants has
an anaplerotic function that replenishes the tricarboxylic acid (TCA) cycle with
organic acids to meet the demands of carbon skeletons for amino acid synthesis
(7). Thus, levels of organic acids other than malate and oxaloacetic acid, as well as
foliar amino acids, would be predicted to increase in the transformants. At present,
only a slight increase in the level of amino acids has been reported in transgenic
potato (23).
Effects of overexpression of PEPC on photosynthesis are controversial. At
temperatures optimal for plant growth, practically no difference in the rate of CO2
assimilation and the CO2 compensation point (0) was observed in transgenic tobacco expressing the maize PEPC gene (25, 31). Activities of PEPC were only
about twofold higher than in wild-type plants in these experiments. In transgenic
rice plants expressing the maize PEPC gene, the rate of CO2 assimilation was also
not altered significantly, but the O2 inhibition of net CO2 assimilation was mitigated with increasing activity of PEPC (32). However, this effect appears not to
have resulted from the fixation of CO2 for photosynthesis by the maize PEPC.
The initial CO2 fixation product, determined by 14CO2 labeling experiments with
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transgenic rice plants having a 50-fold elevation in PEPC activity, was exclusively
the C3 compound 3-phosphoglycerate (13). In addition, overexpression of C4 PEPC
in transgenic rice leaves suppressed CO2 assimilation at 2% O2 to a greater
extent than that at 21% O2, probably through limitation of inorganic phosphate (Pi)
for the Calvin cycle reactions (13). The major increase in PEPC activity may lead
to depletion of Pi in the cytosol, through a stimulation of glycolysis that would
suppresses sucrose synthesis. These collective reactions consume one Pi molecule
and release four Pi molecules, respectively.
Changes in the photosynthetic characteristics at optimal temperatures have been
reported only in transgenic potato expressing the bacterial PEPC (23). In this
case, it was reported that the CO2 compensation point independent of respiration
(0 ∗ ), measured according to Brooks & Farquhar (3), decreased by about 16%
in the transformants with fivefold activity, as compared with wild-type plants
(23). The authors argue that the decrease in 0 ∗ resulted from the increase in
CO2 concentration in the vicinity of Rubisco by decarboxylation of organic acids
produced by PEPC and/or through its anaplerotic function (23). Determination of
the initial CO2 fixation products and quantification of flux through PEPC in these
potato transformants is awaited.
Significant changes in the photosynthetic characteristics in PEPC transformants
have thus far been observed mostly at supraoptimal temperatures. In wild-type
plants, the rate of CO2 assimilation in air declines with increasing temperature,
whereas in PEPC-overexpressing transformants, it remains unchanged or becomes
even greater at higher temperatures in transgenic tobacco (31) and potato (34).
This phenomenon is not yet fully understood, but it might be possible that PEPC
participates in the initial CO2 fixation for photosynthesis (despite the absence
of elevated levels of the other enzymes necessary for a C4 cycle) or that it acts to
increase the CO2 concentration in the vicinity of Rubisco under conditions in which
the oxygenation reaction of Rubisco proceeds much faster that its carboxylation
reaction, as proposed previously (23, 34).
PPDK There are four reports of transgenic C3 plants that express PPDK in the
MC chloroplast (Table 3): transgenic Arabidopsis (27), potato (28), rice (12) expressing the maize C4-specific PPDK gene, and transgenic tobacco expressing a
PPDK gene from the CAM plant Mesembryanthemum crystallinum (44). In all
cases, no changes in photosynthetic characteristics were observed in these transformants, even in the transgenic rice with PPDK activity 40-fold higher than wildtype levels (12). A modest increase in the δ 13C value was reported in the transgenic
potato but this difference was marginal in significance (28). Some changes in the
level and composition of free amino acids were also reported in the transgenic
tobacco (44). In general, the overall PPDK reaction is freely reversible, depending
on concentrations of substrates, activators, and inactivators (5). This is probably
the case in MCs of C3 plants, in which the activity of inorganic pyrophosphatase
and adenylate kinase is low (22) and could be the reason why the overexpression
of PPDK does not result in significant effects on carbon metabolism in C3 leaves.
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The expression of chloroplast-targeted PPDK increased the number of seeds
per seed capsule and the weight of each seed capsule by about 40% and 20%,
respectively, in transgenic tobacco, with about a 1.5-fold increase in activity
of PPDK in the leaves (44). These effects were not observed in transgenic
tobacco plants that express PPDK in the cytosol (44). The mechanism of the
increase in seed yield by PPDK is obscure at present. One possibility is that
overexpression of PPDK in the chloroplast enhances photosynthesis in organs
surrounding seeds. In seed pods of C3 dicots and spikelets of C3 monocots, enzyme activities associated with the C4 pathway are high and C4-like photosynthesis is operative, contributing significantly to grain filling (see 26, 30). Thus,
it is possible that overexpression of PPDK in the chloroplast enhances C4-like
photosynthesis in organs such as hulls and ears to raise the yield of seeds and
grains.
NADP-ME There are four reports of transgenic C3 plants that express NADP-ME
in the MC chloroplast (Table 3): two sets of transgenic rice plants expressing the
maize C4-specific isoform (46, 48), transgenic rice expressing the rice C3-specific
isoform (48), and transgenic potato expressing the C3-specfic isoform of Flaveria
pringlei (34). The transformants expressing the C3-specific isoform with activities
up to several fold higher than wild-type levels did not show any detectable differences in their growth and photosynthesis (34, 48), whereas those overexpressing
the maize C4-specific isoform showed serious stunting and leaf photobleaching,
due to increased photoinhibition of photosynthesis under natural light conditions
(46, 48). It is proposed that the maize C4 NADP-ME in the chloroplasts acts to
increase the NADPH/NADP ratio and to suppress photorespiration, rendering photosynthesis more susceptible to photoinhibition (46, 48). Such detrimental effects
of the maize enzyme might imply significant flexibility of carbon metabolism in
MCs of C3 plants, especially in terms of transport of metabolites between the
cytosol and the chloroplast stroma.
PEP-CK There is only one report of transgenic rice plants that express the C4specific PEP-CK of U. panicoides in the MC chloroplast (45a) (Table 3). Although
this enzyme is located in the BSC cytosol of U. panicoides, the introduced construct was designed so that the enzyme was targeted to the MC chloroplasts in
transgenic rice leaves. The expression of chloroplast-targeted PEP-CK showed
significant alterations of carbon metabolism in rice leaves (45a). In 14CO2 pulsechase experiments of transgenic rice with PEP-CK activity comparable to that in
U. panicoides, about 20% of the radioactivity was incorporated in the C4 compounds, malate, OAA, and aspartate. Feeding of 14C-labeled malate also increased
the incorporation of the radioactivity into sucrose. It is unclear whether the introduced PEP-CK together with endogenous PEPC could drive a C4-like pathway in
MCs of rice leaves as proposed previously, since the CO2 compensation point was
unchanged in transgenic rice plants (45a).
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FUTURE PERSPECTIVES
There has been considerable progress in recent years in the molecular engineering
of C4 photosynthesis. The technology to express the C4 enzymes at high levels and
in the desired locations in the leaves of C3 species is becoming well established,
and it is now possible to produce transgenic C3 plants that express at least a set
of key enzymes of the C4 pathway. Thus, we have just reached the starting point
in introducing the basic biochemical elements of the C4 pathway into C3 plants.
Apart from the goal of installation of a complete C4 pathway into C3 plants, some
transgenic C3 plants that overproduce a single C4 enzyme show alterations in
carbon metabolism. These plants are also proving to be useful tools in probing the
“housekeeping” function(s) of the C4-like enzymes in C3 plants and the evolution
of the C4 photosynthetic genes. Experiments with transgenic plants have reinforced
the fact that the C4 mechanism is a finely tuned metabolic “machine” where both
a high degree of precision in gene expression and structural morphology work
together to concentrate CO2 efficiently at the site of Rubisco. Work with transgenic
C4 Flaveria and also transgenic C3 plants shows that relatively small changes in
leaf biochemistry, induced by transgene action, can have major deleterious effects
on photosynthetic competence. In light of these observations, some important
questions must be answered in the quest to introduce the C4 pathway in C3 crop
plants. First, can we deliver the degree of precision required to coexpress the
necessary genes at the correct levels and ratios in the correct compartments? For
the primary enzymes of the C4 pathway these preliminary results are promising
but correct posttranslational regulation of the introduced, heterologous enzymes,
fine-tuning of the levels of ancillary enzymes (such as CA, adenylate kinase,
and pyrophosphatase) and metabolite transporters must also be addressed. Most
important, can we create an efficient CO2 concentrating mechanism in a plant
lacking Kranz leaf anatomy, a morphological feature independently arrived at
several times through the convergent evolution of C4 plants? Would the metabolic
cost of establishing a CO2 concentrating mechanism without an effective barrier
to CO2 diffusion outweigh the advantages? In connection with this key issue, there
are good examples of higher plant CO2 concentrating mechanisms without Kranz
anatomy, namely, the submersed aquatic macrophytes (SAMs) such as Hydrilla
verticillata, in which an intracellular C4-like pathway is induced in response to a
decline of ambient CO2 concentration (see 36). It remains to be seen, however,
whether this process is a highly efficient addition to the C3 process or a lowefficiency survival mechanism in SAM species. Studies on the mechanisms of
induction of a C4-like pathway in SAM plants may help us to understand how
to introduce an effective C4-like mechanism to MCs of C3 plants. However, a
conclusive answer as to the performance of an artificially introduced C4 pathway
in C3 crops can only be obtained by the generation and comprehensive analysis
of transgenic crop plants such as those described here and those currently being
produced.
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ACKNOWLEDGMENTS
The authors are grateful to Drs RE Häusler, HJ Hirsch, H Honda, RC Leegood,
WC Taylor, and S Toki for providing unpublished information and to Ms Hiroko
Tsuchida for her assistance in preparing the manuscript. Work in the authors’ laboratories was supported by a PROBRAIN grant from the Bio-Oriented Technology
Research Advancement Institution (BRAIN) of Japan and from the Australia/Japan
Bilateral Science Agreement.
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