Biologia 68/4: 577—586, 2013
Section Botany
DOI: 10.2478/s11756-013-0191-5
Review
Evolutionary transition from C3 to C4 photosynthesis
and the route to C4 rice
Zheng Liu1*, Ning Sun2, Shangjun Yang1, Yanhong Zhao3, Xiaoqin Wang4, Xingyu Hao5
& Zhijun Qiao6
1
College of Life Sciences, Hebei University, Baoding, 071002 China; email: [email protected]
The Affiliated School of Hebei Baoding Normal, Baoding, 071000 China
3
School of Agriculture, Ludong University, Yantai, 264025 China
4
College of Biological Science and Engineering, Beijing University of Agriculture, Beijing, 102206 China
5
College of Agriculture, Shanxi Agricultural University, Taigu, 030801 China
6
Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture, Taiyuan,
030031 China
2
Abstract: Compared with C3 plants, C4 plants possess a mechanism to concentrate CO2 around the ribulose-1,5bisphosphate carboxylase/oxygenase in chloroplasts of bundle sheath cells so that the carboxylation reaction work at a
much more efficient rate, thereby substantially eliminate the oxygenation reaction and the resulting photorespiration. It
is observed that C4 photosynthesis is more efficient than C3 photosynthesis under conditions of low atmospheric CO2 ,
heat, drought and salinity, suggesting that these factors are the important drivers to promote C4 evolution. Although C4
evolution took over 66 times independently, it is hypothesized that it shared the following evolutionary trajectory: 1) gene
duplication followed by neofunctionalization; 2) anatomical and ultrastructral changes of leaf architecture to improve the
hydraulic systems; 3) establishment of two-celled photorespiratory pump; 4) addition of transport system; 5) co-option
of the duplicated genes into C4 pathway and adaptive changes of C4 enzymes. Based on our current understanding on
C4 evolution, several strategies for engineering C4 rice have been proposed to increase both photosynthetic efficiency and
yield significantly in order to avoid international food crisis in the future, especially in the developing countries. Here we
summarize the latest progresses on the studies of C4 evolution and discuss the strategies to introduce two-celled C4 pathway
into rice.
Key words: C3 photosynthesis; C4 photosynthesis; C4 rice; hydraulic conductance; photorespiration; plant evolution
Introduction
Photosynthesis is the fundamental biological process
that converts light energy to chemical energy by fixing CO2 into sugars for activities of photosynthetic organisms such as plant, algae and many species of bacteria. However, the first enzyme ribulose 1,5-bisphosphate
carboxylase/oxygenase (Rubisco) responsible for this
process is dual functional, catalyzing the fixation of
both CO2 and O2 . The subsequent biochemical reaction
cycle following CO2 fixation leads to the production
of glyceraldehyde-3-phosphate (G3P) and the process
is called Calvin-Benson cycle (or C3 photosynthesis),
whereas O2 fixation results in the production of toxic
2-phosphoglycolate (2-PG), which should be metabolized by photorespiration at the cost of energy and of
the loss of CO2 that has already been fixed. Rubisco was
proposed to be evolved in a CO2 -rich and nearly O2 free atmosphere about 3 billion years ago, hence selection pressure might have little effect on Rubisco to dis-
criminate O2 from CO2 (Peterhansel et al. 2010). However, significant reduction of atmospheric CO2 /O2 ratio over about 30 million years ago (mya) may have depressed the efficiency and rate of carbon uptake in many
terrestrial plants (Sage 2004). Currently photorespiration causes about 20% loss of net-photosynthesis in C3
plant under moderate conditions and can be even higher
in dry and warm environments (Cegelski & Schaefer
2006). In contrast, photorespiration is almost nonexistent in C4 plants in which CO2 concentrating mechanism helps to eliminate effectively the oxygenation reaction. C4 crops (e.g. maize, sorghum and sugarcane)
are therefore the most productive and resource-use efficient plants exploited by humanity. C4 photosynthesis
involves a complex set of alterations to the anatomy,
biochemistry and cell biology of leaves in comparison
to that of C3 plants. In C4 plants, mesophyll (M) and
bundle sheath (BS) cells surround the vascular tissue in
concentric rings (i.e. Kranz anatomy). Spatial separation of cell-types allows atmospheric CO2 to be initially
* Corresponding author
c 2013 Institute of Botany, Slovak Academy of Sciences
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hydrated to bicarbonate by carbonic anhydrase (CA)
in M cells and then fixed by phosphoenol pyruvate carboxylase (PEPC) to generate four-carbon compound
oxaloacetate (OAA). The later is then converted to
malate or aspartate and is subsequently transported
from M cells to BS cells via abundant plasmodesmata.
The C4 organic acid is decarboxylated to provide high
concentrations of CO2 to Rubisco in BS cells. Finally,
the pyruvate resulting from the decarboxylation reaction is catalyzed by pyruvate, orthophosphate dikinase
(PPDK) in M cells to generate phosphoenol pyruvate
(PEP), the initial substrate of the C4 cycle. In addition
to these, other significant changes in C4 plants include
higher vein density in leaves, increased number and size
of chloroplasts in BS cells and more abundant plasmodesmata linking BS and M cells (Sage et al. 2012).
It is believed that C4 photosynthesis evolved at
least 66 times independently in 19 different families
of angiosperms from C3 ancestors and only relatively
small evolutionary changes were required for the establishment of this photosynthetic pathway (Sage et al.
2011a). Furthermore, it was convinced that during C4
development, C3 state develops by default and serves as
a platform for the establishment of C4 pattern (Langdale 2011), suggesting that the transition from C3 to
C4 is relatively simple. Hence it is rational to significantly increase photosynthetic efficiency in C3 crops
by installing C4 pathway. Rice is the most important
staple crop in the world; however, it operates C3 photosynthesis only. It is estimated that rice yield would
be increased by at least 50% if C4 photosynthesis could
be functional in rice (Sheehy & Mitchell 2011). As it is
going to be challenging to feed the world unless the current yield of crops can be significantly increased in the
next 40 years, understanding why and how C4 evolved
would chart new pathways to create C4 rice.
Ecological drivers for the evolution of C4 photosynthesis
There are 66–68 distinct lineages containing about 7500
species which use C4 photosynthesis and nearly 2/3 of
which (about 4600 species) are in the category of grasses
(Sage et al. 2012). Interestingly, all these lineages share
the initial two steps of C4 pathways (i.e. hydration of
atmospheric CO2 to HCO−
3 and PEP carboxylation);
however, the subsequent steps are unique in different
species to some extent (Langdale 2011). It is also found
that many known lineages occur in clusters. C4 evolution is thus considered to follow the same sequence at
the early steps (Christin et al. 2010). Given that each of
the lineages arose from C3 ancestors as shown by phylogenetic analyses, the subsequent evolutionary paths
are therefore independent in each lineage (Sage 2004).
Thus C4 evolution is proposed to be a combination of
both convergent and parallel evolution (Williams et al.
2012). Although C4 pathways are diverse, the common
concentrating mechanism of CO2 in C4 plants and their
higher photosynthetic characteristics in warm arid conditions supported the ideas that low CO2 , high temper-
Z. Liu et al.
ature, seasonal drought and salinity are main ecological
drivers for the origins of C4 photosynthesis.
Low atmospheric CO2
Atmospheric CO2 is estimated to be over 1000 ppm between 35 and 55 mya, and then it fell abruptly to about
390 ppm during Oligocene by approximately 32-25 mya
(Tipple & Pagani 2007). This dramatic depletion of
CO2 is correlated with the first transition from C3 to
C4 photosynthesis in the grass subfamily Chloridoideae
during the mid-Oligocene epoch, as revealed by molecular clock analysis of the DNA sequences from phylogenetic studies. A novel maximum likelihood approach
to test the link between CO2 decrease and transition to
C4 photosynthetic also supports that Oligocene CO2
decline promoted the establishment of C4 photosynthesis system (Christin et al. 2008). As CO2 is the
basic “food” essentiall to all plants, the more of it
there is in the air, the better they grow. As the atmospheric CO2 content declines, plant growth rates will
decline, especially for C3 plants due to high photorespiration. When a critically-low CO2 concentration is
ultimately reached, starving C3 plants without sufficient CO2 would have difficulty completing their life
cycle during a growing season. Selection then favors C3
to C4 transition for acquisition of carbon-conservation
traits to improve the carbon balance of the plant.
High temperature, drought and salinity
The hypothesis that high temperature is one of the selection factors for C4 photosynthesis was based on the
observation that there is an increased abundance of C4
species along temperature gradients from high to low
latitude and altitude (Teeri & Stowe 1976; Rundel 1980)
and the facts that C4 grasses contribute to over 90% of
the biomass of grasslands at low latitude and altitude
(Tieszen et al. 1997; Wan & Sage 2001). The advantage of C4 photosynthesis over C3 photosynthesis is obvious at high temperature. For example, C3 plants suffer high rate of photorespiration at temperature above
30 ◦C at current atmospheric CO2 levels, whereas photosynthesis of C4 plants performs well with negligible
levels of photorespiration at even higher temperature
(Ehleringer 2005).
Drought and salinity have also been proposed to
influence the evolution of C4 photosynthesis from at
least two aspects: first, drought and salinity (physiological drought) induced the partial closure of stomata which restricted the CO2 supply to photosynthesis
and raised leaf temperature due to decrease in transpiration, resulting in high photorespiration, thus favored selection for CO2 concentrating mechanism of C4
plants (Ehleringer & Monson 1993); second, due to limited supply of freshwater under these stresses, C4 plants
were more competitive than C3 plants because of their
water-conserving mechanism (Brown 1999). Low atmospheric CO2 combined with high temperature, seasonal
drought and salinity caused excessive demand for water transport, resulting in higher stomatal conductance
and increased stomatal opening. C4 photosynthesis fulUnauthenticated
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C4 evolution and C4 rice
filled the high water use efficiency by allowing high
rates of carbon-fixation at low stomatal conductance,
even at low atmospheric CO2 . The resultant decrease in
transpiration allowed stomata to keep open and photosynthesis to be sustained for longer under drought and
salinity conditions (Osborne & Sack 2012).
As low CO2 , elevated temperature, drought and
salinity all promote photorespiration, it has long been
hypothesized that high photorespiration was the primary driver of C4 evolution (Sage et al. 2012). Recently,
hydraulic innovation of plants was also proposed to
be another impetus for C4 evolution (Osborne & Sack
2012). Thus the appearance of C4 plants would be the
result of ameliorated changes in both photorespiration
and hydraulic system promoted by a combination of all
those environmental factors mentioned above.
Evolutionary pathway from C3 to C4 photosynthesis
Evolution of C4 plants may not be initially directed towards C4 photosynthesis, but a gradual improvement
of fitness towards the environmental changes of habitats. C4 pathway emerged in a series of stepwise sequence, each step produced some traits which were evolutionarily beneficial and some early steps were preconditioning for subsequent developments (Gowik & Westhoff 2011). These are in line with the observation that
many variations in the Kranz anatomy and biochemical
pathways exist among C4 species (Edwards & Voznesenskaya 2011). Although diverse in leaf structure and
biochemistry, evolution of C4 photosynthesis underwent
the following transitions in general:
Gene Duplication
Gene duplication refers to the process by which a chromosome, a segment of chromosome or a single gene is
duplicated, resulting in an additional copy of the gene.
It may occur through unequal crossing over, tandem duplication of linked genes, or other illegitimate chromosomal rearrangements. Gene duplication has been considered as one of the principal mechanisms of the evolution of genes with new functions. After a gene is duplicated, the once-identical genes can undergo changes
and diverge to create two different genes. A duplicated
gene may experience advantageous mutations to acquire new function (i.e. neofunctionalization), or subdivide the functions of its progenitor with the other duplicated one through either differentiation of promoters
that regulate different temporal or spatial expressions
or differentiation of functional domains of a protein
(i.e. subfunctionalization) (Monson 2003; Wang et al.
2009). Given that genes encoding enzymes for C4 cycle
often belong to gene families of multiple copies, duplications of whole genomes, genome segments, or only single genes have been considered to be preconditioning for
evolution of C4 photosynthesis (Monson 2003). When
one copy of the gene was evolutionarily modified to obtain a new function to increase the individual fitness
towards a changing environment, the other copy could
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still maintain the ancestral function without producing deleterious consequences. C3 ancestors of C4 genes,
such as CA, PEPC, PEPC kinase (PEPCK), NADPmalic enzyme (NADP-ME) and NADP malate dehydrogenase (NADP-MDH) have been reported to experience duplication, neofunctionalization and/or subfunctionalization events before they were recruited into C4
pathway (Wang et al. 2009; Christin & Besnard 2009;
Ludwig 2012; Rondeau et al. 2005). For example, in C3 ,
C3 -C4 intermediate and C4 species of Flaveria, there
are two separate genes of CA, with its chloroplastic
form containing codes for a functional chloroplast transit peptide and the cytosolic one lacking a functional
transit peptide. It is hypothesized that the chloroplastic copy is ancestral and the cytosolic one appeared
following duplication and neofunctionalization in which
mutations rendered the chloroplastic transit peptide sequence ineffectual. During C4 evolution, the cytosolic
one was then co-opted into C4 cycle by upregulating its
expression in cytosol of M cells, whereas the expression
of chloroplastic one was downregulated (Monson 2003;
Ludwig 2012).
Ameliorating water transport system within leaves
Evolution of increased venation in C3 leaves is thought
to improve hydraulic system initially. But it was a crucial preconditioning step for the establishment of efficient metabolite fluxes of C4 pathway as shorter interveinal distance reduces the path length from M to BS
cells (Gowik & Westhoff 2011). An anatomical study of
54 species containing 21 C3 and 33 C4 eudicots showed
no significant differences in vein density between closely
related C3 and C4 species, indicating that C3 lineages
ancestral to C4 lineages had already obtained high vein
density (Muhaidat 2007). Phylogenetic comparisons of
C3 , C4 and C3 -C4 intermediates of Flaveria also suggest that increased venation evolved earlier than the
development of the Kranz anatomy (McKown & Dengler 2007). It is observed that C3 species from low latitudes and arid areas often have low inter-veinal distances (Sage 2004) and that high solar irradiance and
low humidity in dry environments favor enhancement
of vein density (Roth-Nebelsick et al. 2001). Therefore, increased venation is proposed to be the adaptation to warm and dry climates in C3 progenitors.
In order to keep high rate of photosynthesis to maintain plant growth under drought and warm conditions,
stomata needed to keep open to absorb enough carbon dioxide from the air. However, the longer stomata kept open, the more water they lost. When the
concentration of atmospheric CO2 declined, the water loss through stomata would be further exacerbated.
The plants then evolved more complicated leaf venation systems, to deliver water throughout the leaf more
efficiently. The property of high vein density reduced
the transport distance and resistance for water flow
to stomata where evaporation occurs, thus permitting
longer periods of gas exchange through stomata and
keeping photosynthesis working at the maximal rate.
Developmental studies also showed that establishment
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of vein patterning is an earlier event than formation of
the Kranz anatomy in C4 plants as veins direct differentiation of adjacent cells to form C4 -type photosynthetic
tissues (Langdale 2011; Langdale & Nelson 1991).
Improving water use efficiency and photosynthesis
within single BS cells
Recently it was recognized that in C3 leaves, BS acts
not only as a reservoir of water to cope with a sudden
‘evaporative surge’, but also as a ‘smart pipe’ in controlling water fluxes from the xylem to M cells (Griffiths
et al. 2012). When water leaves the xylem, it enters
BS through aquaporins located in BS membrane, and
subsequently diffuses into M through abundant plasmodestama connected between BS and M. Thus the
aquaporins of BS function as ‘control valve’ of water
transport to dynamically control the leaf hydraulic conductance. Under drought stress, the increased concentration of abscisic acid (ABA) in xylem induces the
downregulation of the BS aquaporin activity in order
to temporarily block the vasculature-mesophyll water
pathway, resulting in the reduction of water flow into
the leaf (Shatil-Cohen et al. 2011; Ache et al. 2010). Selection for increased BS volume allows it to store more
water, buffer water supply to mesophyll and refix respiratory CO2 (Griffiths et al. 2012).
Typically, the photosynthetic efficiency of the BS
is low in C3 plants because the BS contains limited
number of organelles. When establishing an increased
venation with enlarged BS cells, the overall photosynthetic activity of a leaf would drop due to decrease in
volume and numbers of M cells. The evolutionary pressure would favor an increase in the number of chloroplasts as well as the enhancement of Rubisco expression in BS to maintain the overall photosynthetic activity. Photorespiration elevated by enhanced Rubisco activity then required metabolizing the photorespiratory
glycine in single BS cell, correspondingly, the number
of mitochondria and peroxisomes in BS cells increased
(Osborne & Sack 2012; Gowik & Westhoff 2011). The
term ‘proto-Kranz anatomy’ was therefore coined to describe these traits found in C3 species that are closely
related to C3 -C4 intermediates (Muhaidat et al. 2011).
Evolution of the two-celled photorespiration and establishment of a limited C4 cycle between M and BS cells
Once proto-Kranz anatomy was established, the next
step was the evolution of two-celled photorespiration
from single-cell glycine shuttle, an important intermediate stage prior to the establishment of C4 photosynthesis. The initial step for the evolution of two-celled
glycine shuttle may involve the localization of the enzyme glycine decarboxylase (GDC) into the BS tissue.
This may be achieved through molecular manipulation
by the following processes, firstly, duplication of a gene
coding for one subunit of GDC followed by subfunctionalization of two copies expressing separately in the
M and BS; secondly, loss of the expression in M cells
by mutation of the M-specific GDC gene. Hence, the
GDC exhibits active only in BS cells (Sage 2004; Mon-
Z. Liu et al.
son 1999). For example, in C3 -C4 intermediate Moricandia arvensis, the P subunit of GDC became nonfunctional in M cells (Rawsthorne et al. 1988; Morgan
et al. 1993). In order to prevent accumulation of toxic
photorespiratory intermediates in M cells, glycine had
to be transferred to BS cells and then was metabolized
to serine and CO2 by GDC in BS. The liberated CO2
was subsequently refixed by Rubisco in this compartment. Thus, the plants can operate a weak CO2 concentrating mechanism to partially offset deleterious effects
of high photorespiration (Sage et al. 2012). It is observed that extant C3 -C4 intermediates carry out photorespiration between M and BS cells through glycine
shuttle (Bauwe 2010). The leaf anatomy and physiology were then further optimized for a higher efficiency
of photorespiratory CO2 concentrating mechanism. As
a consequence, the Kranz anatomy was formed in which
enlarged BS cells surrounded each vein and the BS cells
were in turn surrounded by M cells. Within BS cells,
organelle numbers were further increased (Muhaidat et
al. 2011; Marshall et al. 2007; Sage et al. 2011b; Voznesenskaya et al. 2007). To re-utilize the photorespiratory
CO2 escaping from BS into M cells, the expression level
of PEPC in the cytosol of the M cells were greatly enhanced. The resulting C4 acids then diffused back to
BS cells for re-assimilation, paving the way for the evolution of true C4 photosynthesis (Sage 2004, Monson
1999). The significant increase in the PEPC activities
were well documented in C3 -C4 intermediates of Flaveria, ranging from five to 20 fold greater than that of C3
species (Monson & Moore 1989). Correspondingly, the
activities of PPDK and NADP-ME were increased in
some C3 -C4 intermediates as compared to that in C3
species, indicating a minimal C4 cycle was conducted
following the establishment of two-celled photorespiratory CO2 pump (Ku et al. 1983; Ku et al. 1991). However, C3 cycle still contributed mainly to overall carbon
gain as evidenced by high Rubisco activity in M cells
(Moore et al. 1988).
Addition of metabolite transport processes
In C3 plants, chloroplasts possess a complete Calvin
cycle and it only requires one transport process across
the chloroplast envelope for every three molecules of
CO2 to be assimilated, i.e. triose phosphate (TP), the
principle product of the Calvin cycle, leaves the chloroplast via the triose phosphate translocator (TPT) in exchange for otho-phoaphate. In C4 plants, however, CO2
assimilation is completed over two distinct cell types,
M and BS cells, requiring the high flux of metabolites
between these two cells to sustain C4 photosynthesis.
For example, in NADP-ME subtype of C4 plants, four
transport processes are required for crossing chloroplast envelope of M cells (i.e. import of pyruvate and
OAA, and export of PEP and malate) and two processes for crossing chloroplast envelope of BS cells (i.e.
import of malate and export of pyruvate) (Weber &
von Caemmerer 2010). Currently, three types of transporters have been identified in C4 plants and they are
PEP/phosphate translocator (Bräutigam et al. 2008),
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C4 evolution and C4 rice
putative OAA/malate antiporter (dicarboxylate transporter) (Taniguchi et al. 2004; Majeran et al. 2008)
and putative pyruvate transporter (Furumoto et al.
2011). Comparative quantitative transcriptomics and
proteomics revealed the significant increase in the abundance of transporters in C4 plants when compared with
that in C3 plants (Bräutigam & Weber 2011). It is
thought that during the evolution of two-celled photorespiration in C3 -C4 intermediates, limited transport
capacity constrained the rapid trafficking of photorespiratory metabolites between M and BS cells. Hence, the
efficient transport networks by increasing the amount
of transport proteins were established to cope with increased flux of metabolites and were later co-opted by
the C4 cycle (Sage & Sage 2008).
Recruitment of C3 genes into a full and functional C4
pathway and adaptive changes of C4 enzymes
Modifications for PEPC M-specific expression occurred
by multiple mechanisms at transcriptional levels, such
as DNA methylation, cis-elements modification, etc. In
maize, demethylation of a PvuII site 3.5 kb upstream
of PEPC is important for M-specific accumulation of
PEPC mRNA (Langdale et al. 1991). However, in C4
species of Flaveria, a 41-bp region of the mesophyll
enhancing module 1 (MEM1) within the 2184-bp promoter of PEPC is necessary for PEPC accumulation
in M cells (Gowik et al. 2004). These reflect the independent but convergent evolution of C4 -type PEPCs in
different species during the early stage when a C4 cycle was established. Evolution of C4 -type PEPCs also
involved the modifications of their kinetic properties,
which happened late in the evolution of C4 photosynthesis, probably in the final stages of C4 evolution (Sage
2004; Sage et al. 2012). After C4 cycle was established,
malate concentrations were substantially increased in
M cells to guarantee its rapid diffuse into BS. However, malate inhibits C3 -type PEPC activity. Therefore it requires reduced PEPC sensitivity to malate.
By comparing PEPC of C4 Flaveria trinervia Mohr
with its ortholog from the closely related C3 Flaveria
pringlei Gand, determinants for high malate tolerance
were identified in the region 2 (aa 297–437) and 5 (aa
646–966) of the enzyme (Jacobs et al. 2008). In addition, C4 PEPC has lower affinity to PEP and sitedirected mutagenesis by substituting serine for alanine
at position 774 of the C3 isoform resulted in its affinity
to PEP close to the C4 enzyme (Bläsing et al. 2000).
These suggest that slight alterations of amino acid sequences of the C3 isoform of PEPC were sufficient for
its adaptation to the new C4 cellular environment during evolution.
As PEPC activity increased through transcriptional regulation in M cells, it competed with Rubisco
of M cells for CO2 . In order to enhance the C4 cycle, the
next step was to compartmentalize Rubisco in chloroplasts of BS cells. The cell specific expression of Rubisco
is determined by the gene of small subunit of Rubisco,
rbcS at both transcriptional and post-transcriptional
levels. In maize, BS specific expression of Rubisco is
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controlled by the region of ZmrbcSm3 from −211bp to
+434bp with respect to the transcriptional start site,
and the regions −885 to −230 and −588 to −182 from
the transcriptional start sites of the ZmRbcSm1 and
ZmRbcSm3 genes, respectively, are responsible for the
absence of M expression (Hibberd & Covshoff 2010).
C3 rice lacks these elements, conferring rbcS expression
in both M and BS cells. Transgenic rice by adding the
maize cis-regulatory elements to the rice promoter region, however, showed BS specific expression of RbsC
(Sage 2004), suggesting that slight alteration of the promoter sequences was sufficient for the transition of RbcS
gene from C3 to C4 cell specific expression. In F. bidentis, BS specific accumulation of RbcS is also thought to
be controlled at posttranscriptional level. Stability determinants located in the 5’ and 3’ UTRs of the transcript determined the posttranscriptional degradation
in M cells (Patel et al. 2006). Cell specific expression of
Rubisco occurred after the limited C4 cycle was established as evidenced that significant decrease of Rubisco
activity in M cells was observed in C4 -like F. brownii,
whereas not in less C4 -like intermediates of Flaveria
species (Reed & Chollet 1985; Cheng et al. 1988). Evolution of Rubisco kinetics occurred late, probably after
the full C4 cycle has been assembled. Two amino acid
substitutions in the Rubisco large subunit at positions
309 and 149 are thought to be associated with the high
catalytic rate in all C4 Flaveria species investigated. In
contrast, no substitutions of these two positions were
found in the C3 species and only one substitution in
the C4 -like F. palmeri (Sage et al. 2012; Kapralov et al.
2011).
Cell-specific expression of C4 -acid decarboxylases
is also important for a full and functional C4 cycle. In
C4 F. bidentis, promoter sequences, coding region and
downstream of the gene NADP-ME are required to generate its BS-specific expression (Ali & Taylor 2001; Lai
et al. 2002; Marshall et al. 2004). In C4 Cleome gynandra, 240 nucleotides in the coding region of NAD-ME directed BS-specific expression of the reporter gene GUS.
Interestingly, this same region derived from NAD-ME
of A. thaliana was sufficient to lead to BS-specific accumulation of GUS in C. gynandra and maize (Brown
et al. 2011). This suggests that genes from C3 species
can be directly co-opted into C4 pathway without any
sequence alteration and indicates that modification of
trans-factors which interact with such conversed ciselement may be required for cell specificity during C4
evolution. Evolution of C4 -acid decarboxylases may also
involve the modifications of structural and kinetic properties. Although such studies are still in their infancy,
it is clear that in maize transition from non-C4 -NADPME to C4 -NADP-ME isoform involved the assembly
of tetramer and increased sensitivity to malate. The
C4 -NADP-ME assembles as a tetramer, whereas the
non-C4 -NADP-ME as a dimer. The region flanked by
amino acid residues 102 and 247 of the C4 isoform was
identified to be critical for its tetrameric state. On the
other hand, pH dependent inhibition of C4 -NADP-ME
by malate which was important to adapt the enzyme
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to a new C4 metabolic context was associated with the
region from residue 248 to the C-terminal end of the C4
isoform (Detarsio et al. 2007).
C4 isoform of PPDK shows high M-specific activity in C4 plant which is necessary to provide sufficient
PEP for the initial generation of C4 acid. By comparing it with its C3 isoform, it was found that the catalytic residues and regions are strictly conserved between the C3 and C4 isoforms and the kinetic properties
for these two functional enzymes are nearly the same
except for their different affinities for pyruvate (Miyao
2003; Chastain et al. 2011). Thus only minor modifications on enzyme properties were required during the
C3 to C4 evolution of PPDK and the major changes
responsible for the evolutionary transition occurred on
the promoter region. In C3 and C4 plants, there exists a conversed arrangement that two promoters control one PPDK gene, giving rise to two transcripts. The
longer transcript encodes a protein containing a chloroplast transit peptide and the corresponding PPDK enzyme accumulates specifically in chloroplasts of M cells,
whereas the shorter one lacks this transit sequence and
the protein generated is cytosolic (Chastain et al. 2011).
As in C3 plants the chloroplastic enzyme expresses only
in low abundance, it was hypothesized that during C4
evolution M-specific enhancing elements were inserted
into a specific region of the promoter which is in charge
of generating the longer transcript, resulting in high
accumulation of the enzyme in C4 M cells (Rosche &
Westhoff 1995).
The path to create C4 rice
It is predicted that that world population would reach
9 billion in 2050. Correspondingly, a significant increase
in crop production would be required to meet the demand of increasing population. As the most important
crop in the world, however, C3 rice exhibits low photosynthetic capacity and inefficient use of nitrogen and
water compared with C4 crops. If C4 cycle could be installed into rice, more than 50% increase in the yield potential would be anticipated (Sheehy & Mitchell 2011).
A large endeavor has been made to produce transgenic
rice overproducing genes specific for C4 pathway in order to enhance to photosynthetic efficiency, but none
of the approaches so far gave satisfactory results, some
even showing deleterious effects to the plants (Miyao et
al. 2011). Because plants have evolved at least 30 million years for the transition from C3 to C4 photosynthesis, there are significant changes of leaf architecture
and cell biology, and all genes, enzymes and organelles
related to C4 cycle work coordinately in different cell
types or in different compartments within a single cell.
Here we summarize the latest strategies for the creation
of C4 rice by combining approaches of the directed evolution and molecular engineering, rather than simply
overexpressing some C4 specific genes in rice.
To our knowledge, no gene responsible for C4
anatomical and cell biological traits (e.g. increase in
venation, enlarged BS cell size, more proliferation of
Z. Liu et al.
organelles in BS cells and more connectivity of plasmodesmata between M and BS cells, etc) has been
identified till now and the underlying molecular mechanisms remain largely unknown. Thus the directed evolution approach seems to be one choice to acquire these
traits in the shortest possible time. As high photorespiration and water deficit promote C4 evolution, generation of activation-tagged rice populations with the
aid of chlorophyll fluorescence imaging (Sage & Sage
2008) to screen young seedlings showing the C4 traits
under low CO2 , elevated growth temperature and moderate drought have been performed (Cowling & Sage
1998). Currently, only mutants with reduced inter-vein
distances and reduced numbers of M cells between veins
in rice have been successfully identified (Kajala et al.
2011). We should be aware that it is going to be risky
to use these mutants showing only one C4 trait (i.e. increased venation) for further introduction of C4 pathway because an efficient C4 cycle depends on all of
the C4 traits mentioned above. So endeavors should be
made to acquire mutants showing other important C4
traits (Liu & Sun 2012) by generating a larger mutant
population and performing mutagenesis in different varieties of rice species. Once these works bring us success,
the rice mutants showing different C4 traits could then
be crossed to obtain multiple desirable traits in a single plant. Meanwhile, transcriptomic and/or proteomic
comparisons of closely related C3 and C4 species, such
as Flaveria (Gowik et al. 2011), Cleome (Bräutigam et
al. 2011) and some distantly related plants (Bräutigam
et al. 2008), of different developmental stages of C4
leaves (e.g. Hydrilla system) (Liu & Sun 2012), of leaf
developmental gradient of the same leaf (Li et al. 2010;
Majeran et al. 2010) and of M vs. BS of the C4 plants
(Majeran et al. 2005; Majeran et al. 2008; Friso et al.
2010) were or are also being conducted to look for master genes switching the developmental transition to C4
and core suite of genes necessary to maintain C4 operation. These works combining system biology would
accelerate our efforts to create C4 rice.
Previous attempts to engineer single-cell C4 photosynthesis in rice also revealed the importance of intracellular metabolite transport for a closed C4 cycle.
Without any modification of metabolite transport system, the transgenic rice plants only generate futile cycles with little or even no contribution to C4 photosynthesis (Miyao et al. 2011). So one important consideration to produce C4 rice would be the addition
of transporters required for massive fluxes of metabolites importing and exporting organelles in different cell
types. Although some important transporters have been
identified through comparative proteomics of chloroplast envelopes from M and BS cells in maize and from
C3 and C4 plants (Langdale 2011), other transporters
such as those for import of pyruvate into chloroplast in
M cells and all the BS cell-specific transporters remain
unknown. It is crucial to identify these transporters by
functional assays of potential candidates obtained from
the data of comparative ‘omics’ (Bräutigam et al. 2008;
Gowik et al. 2011; Chang et al. 2012).
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C4 evolution and C4 rice
The regulatory and coding sequences of the C4
genes should also be thoroughly characterized and carefully selected before transferring them into rice. As
overexpression of C4 genes in rice driven by 35S promoter often caused unexpected negative results, the
endogenous C4 promoters for cell-specific expressions
of C4 genes should be considered (Kajala et al. 2011).
This strategy seems to be promising for correct expression of M-specific genes in rice. For example, strong Mspecific expression patterns of PEPC and PPDK were
successfully achieved in rice by using their endogenous
C4 promoters from maize respectively (Ku et al. 1999;
Fukayama et al. 2001; Suzuki et al. 2006). However,
it is complicated for BS-specific genes (e.g. RbcS and
NAD(P)-ME) as their cell-specific expression is regulated at post-transcriptional level, probably by transacting factors (Peterhansel 2011). One solution would
be to identify these trans-acting factors from C4 plants
and co-transform them with C4 BS-specific genes to
rice (Peterhansel 2011). It should be mentioned that
C4 specific genes are usually regulated by metabolic
(Sheen 1990; Kausch et al. 2001) and environmental
stimuli (Berry et al. 1990; Danker et al. 2008) or under
tight circadian control (Horst et al. 2009) in C4 plants.
Although these properties are important for the economy of the C4 cycle, they were neglected in the previous
studies for producing C4 rice. In our opinion, synthetic
promoters would be of the best choice. In fact, it was
already demonstrated that the synthetic promoter combining the dehydration responsive element from Arabidopsis with guard cell specific element from potato
successfully drove the expression of the reporter gene
GUS specifically in guard cell of tobacco under drought
condition (Li et al. 2005). Hence, cis-elements responsive to various stimuli or responsible for cell type specificity derived from different species could be combined
to synthesize a novel promoter so that it can mimic the
natural regulatory mechanisms of C4 genes in transgenic rice. Kinetic properties of C4 enzymes are usually regulated by various metabolites in C4 plants. As
different metabolic conditions exist in C3 plants compared with that in C4 plants, engineering the coding sequences of C4 genes to adapt to C3 environment seems
not to be simple. For example, phosphorylation of C4
PEPC, which is important in the control of feedback
inhibition, occurs in the light in maize, whereas it happens in the dark when overexpressing the C4 protein in
rice (Fukayama et al. 2003). In order to install a fully
functional C4 metabolism in rice, it is necessary to test
the regulatory properties of those C4 enzymes under
C3 background to begin with (Peterhansel 2011) and
subsequently to manipulate coding sequences of these
proteins through either site-directed mutagenesis or directed evolution.
Challenges and future perspectives
Increasing data from paleoecology, climatology and
molecular, physiological, developmental and system biology in recent years provide a detailed understand-
583
ing of C4 evolution on the one hand, but on the other
hand generate new evidences challenging the existing
hypotheses. One challenge toward the mainstream hypothesis of C4 origin is that the earliest C4 grasses
would trace back to the Middle Eocene, a time of warm
equable climates and probably of high CO2 (Edwards
et al. 2010). Palaeohistorical studies also showed that
molecular divergence of monocot and dicot C4 lineages
occurred 20-30 mya and C4 grasses dominated in savanna biomes around 5-8 mya (Christin et al. 2011;
Cerling et al. 1997; Ehleringer et al. 1997). However,
only about 2 mya the atmospheric CO2 declined to 280
ppm, thus too late to drive C4 diversification (Griffiths
et al. 2012). Given that C4 plants were likely originated
in seasonally dry, warm habitats, leaf hydraulic conductance rather than CO2 concentration could be considered as the major driver for C4 evolution (Osborne &
Sack 2012; Griffiths et al. 2012). The other hypothesis
to be assessed is that gene duplication and subsequent
neofunctionalization was the initial crucial step for the
evolution of C4 genes. By comparing ten gene families
of known C4 pathway components from three C3 and
three C4 species whose genomes were completely sequenced, none of the C4 plants shows a clear increase
in gene copy number (Williams et al. 2012). Combining the facts that multiple C3 genes possess elements
to direct M or BS specificity in C4 plants (Brown et
al. 2011; Kajala et al. 2012), it implies that C3 othologues of C4 genes had already been predisposed for C4
evolution before they were recruited into the C4 cycle
and gene duplication seems not to be necessary for the
evolution of C4 pathway (Williams et al. 2012). Earlier
emergence of increased venation than that of high BS:M
ratio during C4 evolution is also in debate as proliferation of BS cells was observed to be more advanced than
any increase in vein density in C3 species of PACMAD
lineages, to which all grasses with the C4 pathway are
restricted (Griffiths et al. 2012).
Undoubtedly, understanding C4 evolution will shed
light on our current endeavors to generate C4 rice. The
lessons from engineering a single-cell C4 photosynthetic
pathway into rice (Miyao et al. 2011) make us realize
that there is no shortcut for the successful generation of
C4 rice, but to at least partially follow the sequence of
C4 evolution. So the preliminary work would use strategies of directed evolution to obtain mutant rice lines
anatomically and cell biologically similar to C4 plants
before they are installed with C4 pathway. Other challenges to create C4 rice include that the existing Calvin
cycle will compete for CO2 with the C4 pathway introduced and photorespiration in M cells will result in unnecessary loss of CO2 , hence interfering the operation of
the C4 cycle. Both pathways should be re-orientated to
BS cells through the elimination of their performances
in M cells (Kajala et al. 2011). The existence of chloroplastic PEPC in rice is also of the problem because it is
thought to play a crucial role in ammonium assimilation
under anaerobic conditions (Masumoto et al. 2010),
however, it could create futile cycling of the metabolites when C4 cycle is introduced (Kajala et al. 2011).
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584
It seems that when engineering C4 pathway into rice, establishment of nitrogen assimilation bypass should also
be required simultaneously.
In the next 40 years, there will be an increasing
food demand from the growing population of the world.
‘Supercharging rice’ by introducing C4 photosynthesis
to dramatically increase the yield is of the objective to
be fulfilled in 15 to 20 years because C4 crops are among
the most productive plants on the planet from the aspects of photosynthetic efficiency, harvestable yield and
total biomass. Although this project remains a grand
challenge, advances in modern technology provide great
promise to achieve the goal. We optimistically anticipate that the combination of molecular genetics, genomics, developmental biology, system biology, phylogenetics, plant physiology and ‘omics’ will provide new
insights into C4 photosynthesis and further the efforts
on C4 engineering.
Acknowledgements
We thank the Scientific Research Foundation for the Returned Overseas Chinese Scholars by State Education Ministry of P. R. China (No. 2011-1139), the Talent Introduction Project of Hebei University (No.2010-185), Promotive
Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (BS2009NY014) and the National Natural Science Foundation of China (30800682) for
funding.
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Received December 10, 2012
Accepted January 23, 2013
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