全 文 ：植物生理学报 Plant Physiology Journal 2011, 47 (12): 1127~1136 1127
Received 2011-09-24 Accepted 2011-10-31
* Corresponding author (E-mail: xinguang.zhu@gmail.com; Tel: 86-21-
54920486; Fax: 86-21-54920451).
特约综述 Invited Review
C4 Rice: Are We Ready For The Challenge? A Historical Perspective
LI Yuan-Yuan1,2, ZHANG Hui2,3, ZHU Xin-Guang1,2,*
1State Key Laboratory of Hybrid Rice Research, 2Key Laboratory of Computational Biology and Partner Institute for Computa-
tional Biology, CAS-MPG Partner Institute of Computational Biology, Shanghai Institutes of Biological Sciences, Chinese Acade-
my of Sciences, Shanghai 200031, China; 3Key Laboratory of Plant Stress Research, Shandong Normal University, Jinan 250014,
China
Abstract: The world is entering an age when the increased demand for food production requires substantial en-
hancement of the crop productivity. Ever since the discovery of C4 photosynthesis in the late 1960s, attempts of
engineering the current major staple crops to perform C4 photosynthesis have never stopped. Unfortunately,
relatively little success has been achieved so far, which has created tremendous doubt in research community
and policy makers alike regarding whether it is in the end possible to engineer C3 crops to perform C4 photo-
synthesis. Paramount of evidences with the rapid advances in the next generation sequencing technologies and
new approaches in genetic engineering suggest the C4 engineering is a tangible goal. In this review, we dis-
cussed the rationales behind current major research activities. We begin by summarizing previous genetic stud-
ies through crossing. We further demonstrate that the combination of the next generation sequencing technology
and a new model species for C4 photosynthesis research Setaria viridis will tremendously expedite our discov-
ery of key genes controlling C4 development. Finally, we emphasize that though the C4 engineering has gained
major momentum in scientific research community however its final success will require continued support
from major different bodies, including not only public funding bodies to charity foundations.
Key words: C4 photosynthesis; C4 engineering; genetic engineering; next generation sequencing; Setaria
viridis
C4水稻, 我们的新挑战
李圆圆1,2, 张慧2,3, 朱新广1,2,*
中国科学院上海生命科学研究院1国家杂交水稻重点实验室, 2计算生物学研究所计算生物学重点实验室, 上海200031; 3山
东师范大学逆境植物重点实验室，济南250014
摘要: 自从上个世纪60年代末C4光合途径发现以来，人们对工程改造现有C3粮食作物使之具有C4光合能力进行了大量努
力。目前，大量分子、生理和基因组水平研究的进展和证据表明，该目标将可能在10~15年之内实现。本综述结合目前
国际C4研究的现状，详述了该领域目前所涉各项研究内容的理论依据。我们首先总结过去的经典杂交实验，然后论证新
一代测序技术与C4光合研究模式系统狐尾草(Setaria viridis)的发展极大的促进了我们对C4光合特征遗传发育相关基因的
发现与鉴定。最后，我们强调虽然C4光合工程改造的研究目前已在世界各国大规模展开，但其最终成功仍有赖于不同国
家研究基金及私立慈善基金的大力和长期共同资助。
关键词: C4光合途径; C4光合工程改造; 遗传工程改造; 新一代测序技术; 狐尾草
In C3 plants, CO2 uptake process occurs in the leaf
mesophyll (M) cells where CO2 is catalyzed by ribu-
lose-1,5-bisphosphate carboxylase/oxygenase (Rubis-
co; EC 4.1.1.39) to react with Ribulose bisphosphate
to produce 2 molecules of 2-phosphoglyerate. Rubis-
co is a bifunctional enzyme because it can also cata-
lyze the reaction of ribulose-1,5-bisphosphate (RuBP)
with O2 to generate one molecule of 3-phosphoglyerate
and one molecule of 2-phosphoglycollate, which fur-
ther goes through the photorespiratory pathways and
植物生理学报1128
recycle 3/4 of the carbon back to the Calvin cycle.
Photorespiratory flux can be up to 49% at current O2
and CO2 concentrations and typical Rubisco kinetics
(Zhu et al. 2008). As a result, species evolved means
to reduce photorespiratory fluxes gain a strong evolu-
tion selection advantage, especially under low CO2
and drought conditions which promote photorespira-
tion (Bauwe 2011). C4 photosynthesis represents a
success of evolution, where a biochemical CO2-con-
centrating mechanism formed to elevate the CO2 con-
centration around Rubisco to be up to 10-fold the
concentration in bundle sheath (BS) cells, which ef-
fectively suppress photorespiration under these con-
ditions (Hatch 1987; Furbank 2011).
C4 photosynthesis represent a specialization of bio-
chemical, cellular and anatomical modifications over
C3 plants (Hatch 1987). Unlike C3 photosynthesis
which is accomplished within a single M cell, C4
photosynthesis requires a metabolic cooperation be-
tween two morphologically distinct cell types-BS cell
and M cell. In most C4 plants, CO2 is initially fixed
by phosphoenolpyruvate carboxylase (PEPCase; EC
4.1.1.31) in M cells to form the C4 acid oxaloacetate
(OAA). OAA is subsequently converted to malate in
chloroplasts by NADP-dependent malate dehydroge-
nase (NADP-MDH, EC 1.1.1.82) or to aspartate in
cytosol by aspartate aminotransferase (AST, EC
2.6.1.1). Either malate or aspartate is transferred to
neighboring BS cells, where CO2 is released by de-
carboxylation and then enters the Calvin cycle. Three
different decarboxylation enzymes have been identi-
fied in BS cells of C4 plants, which are NADP-de-
pendent malic enzyme (NADP-ME; EC 1.1.1.40),
NAD-dependent malic enzyme (NAD-ME; EC
1.1.1.39), or phosphoenolpyruvate carboxykinase
(PEPCK; EC 4.1.1.49). C4 photosynthesis has been
further subdivided into three subtypes (NADP-ME,
NAD-ME, PEPCK subtypes) according to the prima-
ry decarboxylation enzyme used to release CO2 in the
bundle sheath. The major C4 crops, e.g. maize, sor-
ghum, sugarcane, Miscanthus, and switchgrass, are
all characterized as NADP-ME subtype, though the
advantage in terms of efficiency has not been charac-
terized (Furbank 2011). In fact, considerable evi-
dence showed that a mixed pathway of decarboxyla-
tion exists in some C4 plants, especially in the
NADP-ME subtype (Calsa and Figueira 2007; Chap-
man and Hatch 1981; Furumoto et al. 1999, 2000;
Hatch 1971; Meister et al. 1996; Wingler et al. 1999)
and Furbank (2011) proposed that flexibility in decar-
boxylation mechanisms may be both developmentally
and environmentally controlled (Fig.1).
One of the major advantages of C4 photosynthesis
to C3 is high photosynthetic energy conversion effi-
ciency. The theoretical maximal energy conversion
efficiency of total solar energy to biomass is 6% in a
C4 plant, while it is around 4.6% in a C3 plant (Zhu
et al. 2008). The other two major advantages of C4
photosynthesis are enhanced water-use efficiency
(WUE) and nitrogen-use efficiency (NUE) (Bolton
and Brown 1980; Huxman and Monson 2003; Li
1993; Morison and Gifford 1983; Sage and Pearcy
1987; Schmitt and Edwards 1981). These high light,
water and nitrogen use efficiencies arouse the strong
interest of plant biologists to engineer C4 photosyn-
thesis into rice (Oryza sativa L.) or other important
C3 crops to improve productivity in order to meet the
food demand of an increasing world population (Hib-
berd and Covshoff 2010; Hibberd et al. 2008; Mat-
suoka et al. 2001; Zhu et al. 2010a). Many recent ad-
vances related to C4 engineering have been
summarized in the special issue of Journal of Experi-
mental Botany ‘Exploiting the engine of C4 photo-
synthesis’ (Volume 62, No 9, 2011), which updates
the recent developments and future promise in vari-
ous areas of C4 biology (Sage and Zhu 2011).
Given the vast number of research areas pertinent to
C4 engineering, it is a major challenge for entry sci-
entists to fully appreciate the rationale behind various
projects related to C4 rice engineering efforts. This
review aims to fill in this gap. Here we start by re-
viewing historical studies of interspecific crossing,
李圆圆等: C4水稻，我们的新挑战 1129
followed by rationalizing the current efforts in using
large scale mutation screening using phenomics facility.
We further explain the major advantages of using the
next generation sequencing technology to identify
key regulators and genes controlling particular mu-
tant phenotype. After further introduction of a new
C4 model system, Setaria viridis, which serves as the
genetic workhorse in support of C4 research, we end
by emphasizing that the success of C4 engineering
will require the continued support from various fund-
ing bodies in different countries.
1 Classic genetic manipulation experiments by
crossing
To date, 62 independent lineages of C4 photosynthe-
sis were identified (Sage et al. 2011). C4 plants were
reported in different families and genera. A few gen-
era contain both C3 and C4 species, such as Atriplex,
Flaveria and Panicum. The attempt of introducing
C4 photosynthesis into C3 plants to reduce photo-
respiration and increase photosynthetic capacity was
initiated in late 1960s through conventional interspe-
cific hybridization between C3 and C4 species within
one genus (Brown and Bouton 1993).
1.1 Interspecific hybridization in Atriplex
The earliest hybridization of photosynthetic types
was done in Atriplex by the Carnegie group (Björk-
man et al. 1969). The most extensive cross in Atriplex
was done between Atriplex rosea (C4, NAD-ME
type) and Atriplex triangularis (C3) (Björkman et al.
1970, 1971; Boynton et al. 1970; Nobs et al. 1970;
Pearcy and Björkman 1970). The F1 hybrids of this
cross had an intermediate leaf anatomy between their
Fig.1 A schematic diagram of a typical NADP-malic enzyme (NADP-ME) type C4 photosynthesis.
The upper graph shows a typical Kranz anatomy. The bundle sheath (BS) cells show thickened cell wall and centrifugally arranged chloroplasts.
The mesophyll (M) cells are closely connected to BS cells at the outside. The basic biochemical reactions invoveld in a coupled BS and M cells
are described. The typical CO2 concentrating mechanism and metabolite transport processes involved in NADP-ME type C4 photosynthesis are
represented in the bottom two coupled BS and M cells. Adapted from Zhu et al (2010b) with permission.
植物生理学报1130
parents with bundle sheaths containing numerous
chloroplasts (Boynton et al. 1970) and PEPcase ac-
tivity was detected in F1 generation plants (Björkman
et al. 1971; Pearcy and Björkman 1970). However,
CO2 compensation point (Г) of these plants was simi-
lar to those of C3 parent and 14CO2 incorporation
studies suggested that no C4 cycle operates in these
plants (Björkman et al. 1970; Pearcy and Björkman
1970). In the F2 generation, there was considerable
variation in Г value, PEPcase activity, and leaf anato-
my, but δ13C (stable carbon isotope ratio) was typical
of C3 values (Björkman et al. 1970; Boynton et al.
1970). In the F3 generation, though segregation of C4
traits were observed, no plant with C4-like photosyn-
thesis was found as in the F2 generation (Oguro et al.
1988). The variable numbers of chromosomes in F2
and F3 generation plants made it difficult to study the
C4 biochemical and leaf-anatomical features in the
successive generations (Björkman et al. 1971).
1.2 Interspecific hybridization in Panicum, Mori-
candia, and Brassica
Brown et al. (1985) performed three inter-specific
crosses in Panicum between C3 and C3-C4 interme-
diate types. All three F1 hybrids between Panicum
milioides Nees ex Trin. (C3-C4) and P. laxum Sw.
(C3), P. spathellosum Doell (C3-C4) and P. bolivi-
ense Hack. (C3), and P. spathellosum and P. laxum
showed leaf anatomical traits (especially organelle
quantities in BS cells) and Г values between those of
their respective parents (Brown et al. 1985). But
these hybrids were sterile and found to have many
unpaired chromosomes (Brown et al. 1985). Colchi-
cine treatment doubled their chromosome number
and restored fertility. Bouton et al. (1986) further
studied CO2 exchange, morphological, and leaf ana-
tomical characteristics of the F2 or F5 generations. In
the segregating F2 progeny, the Г values were typical
of C3, but not of C3-C4 plants and leaf anatomy and
overall plant morphology of some plants showed var-
ious combinations. Variability among F5 generation
plants appeared to be as great as among F2 individu-
als (Bouton et al. 1986).
1.3 Interspecific hybridization in Falveria
Flaveria is a genus with C3, C4, C4-like (incomplete
compartmentation of photosynthetic enzymes be-
tween M Cells and BS Cells), and C3-C4 intermedi-
ate (a partially developed Kranz leaf anatomy and re-
duced levels of photorespiration relative to C3 plants)
species. Many interspecific crosses have been made
between species differing in the degree of C4 photo-
synthesis. Holaday et al. (1985) hybridized Flaveria
pringlei (C3) with F. brownii (C4), the hybrid of
which exhibited reduced Г values. In addition, the
activities of C4 enzymes (PEPcase, PPDK, NADP-
ME) in F1 hybrid plants were higher than in C3 par-
ent, but only 7–10% of those in C4 parent. But F1 hy-
brids still possessed the mesophyll arrangement
characteristic of C3 parent. Hybridization of C4 or
C4-like with C3-C4 or C3 species in Flaveria also
showed that only some C4 traits could be transferred
to C3-C4 species, not the fully developed C4 photo-
synthesis pathway (Brown et al. 1986; Cameron et al.
1989; Holaday et al. 1988).
Although interspecific hybridization could reduce
photorespiration to some extent in some C3 species,
introducing the fully developed C4 photosynthesis
pathway into C3 plants through interspecific hybrid-
ization is far more unrealistic than it was thought.
2 Mutant screening in rice, maize and sorghum
Maize (Zea mays L.), a globally important C4 crop,
has been extensively investigated as a model species
for C4 photosynthesis research. However, up to date,
very little is known about genetic regulation of C4
differentiation. It is largely caused by few results
from large-scale genetic screens for C4 mutants.
Langdale’s group took advantage of transposon-me-
diated mutagenesis to perform a forward genetic
screen in maize for C4 mutants with perturbed leaf
development and identified several mutations that
李圆圆等: C4水稻，我们的新挑战 1131
disrupt bundle sheath cell differentiation (Langdale
and Kidner 1994; Roth et al. 1996). The correspond-
ing mutants were known as bundle sheath defective
(bsd) mutants, which showed cell-specific mutant
phenotypes, with normal mesophyll cells and abnor-
mal bundle sheath cells. The mutants bundle sheath
defective 1 (bsd1, allelic to golden2) (Fitter et al.
2002; Hall et al. 1998; Langdale and Kidner 1994;
Rossini et al. 2001; Waters et al. 2009) and bundle
sheath defective 2 (bsd2) (Brutnell et al. 1999; Roth
et al. 1996; Wostrikoff and Stern 2007) were well
characterized. However, these studies showed that
both genes were not directly regulating C4 differenti-
ation. BSD1 (G2) might co-regulate and synchronize
the expression of a suite of nuclear photosynthetic
genes (Waters et al. 2009). BSD2 is involved in the
assembly of Rubisco holoenzyme (Brutnell et al.
1999) and displays a BS-specific defect because
Rubisco is localized to the BS chloroplast. Therefore,
this screen for mutants exhibiting BS cell-specific de-
fects is likely to identify mutations that indirectly af-
fect compartmentation more than regulators of C4
differentiation.
Recently, with funding from the Bill and Melinda
Gates Foundation, an international consortium of sci-
entists was established to work on the challenge of
engineering a two-cell C4 photosynthesis pathway
into C3 rice which is called C4 rice project (www.
c4rice.irri.org). Part of this project is phenotyping
large populations of rice mutants and wild relatives
for ‘C4-ness’ to identify C3 plants that have acquired
C4 characteristics and screening maize and sorghum
(Sorghum bicolor L.) mutants for loss of C4 function
(Furbank et al. 2009). A variety of plant phenomics
approaches that can be rapidly applied to a large
number of plants were used in these large-scale
screens, including anatomical, biochemical and phys-
iological assays. For instance, screening for vein
spacing in intact C3 and C4 leaves with a hand-held
microscope and screening for gain of C4 function in
rice and loss of C4 function in sorghum by analyses
based on the CO2 compensation point in a home-
made large-volume CO2-controlled chamber (Furbank
et al. 2009). Some interesting mutants with altered
vein spacing have been identified and are undergoing
detailed characterization.
Considering that multiple important regulators of
many signaling pathways were identified by large-
scale genetic screens in mutagenized Arabidopsis
population. Thus, high-throughput genetic screens in
rice or C4 plants will provide valuable information in
defining the networks underlying C4 differentiation.
3 Expression of C4 genes in C3 plants
It is well known that C3 plants lack a C4 carbon
shuttle compare to C4 plants. During the last 20
years, scientists put a lot of efforts in transferring
genes encoding the enzymes of the C4 photosynthetic
shuttle to C3 plants. Though some genes from C4
plants can be correctly expressed in M- or BS-cells in
closely related C3 species, none of these attempts so
far resulted in transgenic C3 plants with enhanced
photosynthesis or growth (Peterhansel 2011). How-
ever, these researches provide valuable information
about the behavior of the introduced genes and in-
form future work.
3.1 Mesophyll specific C4 enzyme transfer
The promoters of C4 genes encoding PEPC, PPDK,
and aspartate aminotransferase (AAT) can drive the
reporter gene specifically expressed in M cells of C3
species. The expression of 5 flanking region of
PEPC (−1 212 to +78) from maize fused to the re-
porter gene encoding β-glucuronidase (GUS) was ex-
pressed almost exclusively in M cells of transgenic
rice and also regulated by light as observed for most
other photosynthetic genes (Matsuoka et al. 1994).
This suggests that regulation of the C4 PEPC expres-
sion is predominantly promoter-controlled; the regu-
lation systems directing cell-specific and light-induc-
ible expression of PEPC in C4 plants also exist in C3
plants (Matsuoka et al. 1994). When the intact maize
植物生理学报1132
C4 PEPC gene containing its promoter, all exons, in-
trons and the terminator was transferred to rice, most
transgenic rice plants showed high-level expression
of active enzyme and exhibited reduced O2 inhibition
of photosynthesis (Bandyopadhyay et al. 2007, Ku et
al. 1999). However, photosynthesis was slightly in-
creased at high temperatures and this may be caused
by the increased stomatal conductance in the trans-
genic rice plants (Bandyopadhyay et al. 2007). When
the intact maize C4 PEPC gene (genomic fragment
with its own promoter and terminator) was intro-
duced into tobacco (a phylogenetically distant C3
species), PEPC gene was not faithfully transcribed as
transcription initiation shifts to upstream of the nor-
mal site though its expression was still regulated by
light (Hudspeth et al. 1992). Similar to PEPC, the
promoter of the maize PPDK gene directed GUS ex-
pression only in M cells and GUS expression was
also regulated by light in transgenic rice plants (No-
mura et al. 2000; Matsuoka et al. 1993). The intact
maize C4 PPDK gene containing its own promoter
and terminator and exon/intron generated high-level
and active PPDK enzyme and functioned better than
chimeric gene containing C4 PPDK coding region
fused to the rice Cab promoter (Fukayama et al.
2001). This indicates that the regulation of C4 PPDK
is also conserved in C3 plants as it is in C4 plants.
The promoter of Panicum miliaceum cytosolic AAT
gene (PcAat) also directed the expression of reporter
gene in cell-specific and light-dependent manner in
the transgenic rice plants (Nomura et al. 2005b).
Therefore, the straightforward strategy to express C4
genes in C3 species is to use endogenous C4 promot-
ers from phylogenetically close C4 species (Peter-
hansel 2011).
3.2 Bundle sheath specific C4 enzyme transfer
The high-efficiency C4 carbon assimilation pathway
also requires BS cell-specific C4 enzymes, like NA-
DP-ME, PEPCK, Rubisco, mitochondrial AAT
(PmAat), glycine decarboxylase (GDC). The expres-
sion of these C4 genes has been tested in C3 plants.
NADP-ME and PEPCK are specifically expressed in
BS cells in NADP-ME-type and PEPCK-type C4
plants, respectively. When the 5 region to maize NA-
DP-ME gene (−1 032 to +463, containing the 5
flanking region, first exon, first intron, and part of the
second exon) fused with GUS reporter gene was in-
troduced into rice, GUS was accumulated not only in
BS cells but also in M cells of transgenic plant leaves
(Nomura et al. 2005a). In contrast, in leaves of trans-
genic rice, the 5 region of the PEPCK gene from
Zoysia japonica (−1 447 to +227, containing the 5
flanking region, first exon, first intron, and part of the
second exon) directed GUS expression in BS cells,
not in M cells (Nomura et al. 2005a). The expression
of GUS reporter gene under the control of P. miliace-
um PmAat promoter showed neither BS cell-
specificity nor light responsiveness when the chime-
ric gene was placed in rice (Nomura et al. 2005b).
Even introducing the intact PmAat gene into rice,
there was still no cell-specific expression pattern ob-
served (Nomura et al. 2005b). The 5 region (−444 to
+66) of maize RbcS gene encoding small subunit of
Rubisco generated GUS accumulation in M cells of
leaf blades and leaf sheaths in a light-inducible man-
ner (Matsuoka et al. 1994). However, the region did
not include all of the elements required for repression
in M cells of maize (Bansal et al. 1992; Hibberd and
Covshoff 2010; Purcell et al. 1995; Viret et al. 1994).
Glycine decarboxylase (GDC) plays an important
role in plant photorespiratory. In C4 species, it is re-
stricted in BS cells. The promoter of the GDCPA
gene encoding GDC P-subunit from F. trinervia di-
rected GUS reporter gene expression in BS cells in
transgenic Arabidopsis plants (Westhoff et al. 2008).
General speaking, it is more difficult for BS cell-spe-
cific C4 enzymes than M cell-specific C4 enzymes
faithfully expressed in C3 species.
3.3 Multiple genes transfer
Efficient C4 photosynthesis requires a high level of
coordination, including high expression of C4 genes,
suppression of a number of genes, introduction of
李圆圆等: C4水稻，我们的新挑战 1133
transporters, anatomical development and so on.
Though it is not necessary to engineer all the related
genes into rice to install a functional C4 cycling, pre-
vious research shows that single C4 gene transfer
simply can’t generate C4 rice. Multiple transgenes
are tested to achieve C4 rice. The most commonly
used method to combine multiple transgenes in a sin-
gle plant is through genetic crossing of individual
transgenic lines. One of our collaborators, Dingyang
Yuan (China National Hybrid Rice R&D Center), has
successfully introduced four C4 genes (PEPC, PPDK,
NADP-ME and NADP-MDH) into rice using con-
ventional hybridization. We are working together to
conduct detailed analysis of these engineered plants.
Peterhansel (2011) reviewed other possible strategies
for multiple integration.
C4 carbon cycling is not the whole story of C4 pho-
tosynthesis and Kranz anatomy is also required for
highly efficient two-celled C4 photosynthesis. But no
genes controlling Kranz anatomy have been identi-
fied so far. Thus, it can be understood that it never
succeed in enhancing the photosynthetic capacity of
transgenic C3 plants through placing C4 genes into
C3 species.
4 High-throughput analysis to search for candi-
date regulators
Recent technological innovations including next-gen-
eration sequencing (NGS) technologies and sophisti-
cated mass spectrometry instrumentation greatly ben-
efit plant science research. Based on NGS, RNA-Seq
is a recently developed technology that allows the
entire transcriptome to be surveyed in a very high-
throughput and quantitative manner (Wang et al.
2009). With RNA-seq, it is possible to examine the
transcriptome of a species without knowing its ge-
nome sequence, such as C4 species, Cleome, Flaver-
ia and produce its transcriptome database (Bräutigam
et al. 2011b). RNA-seq analysis of the closely related
species C3 (Cleome spinosa) and C4 (Cleome gynan-
dra) showed up to 603 transcripts differed in abun-
dance between these C3 and C4 mature fully differ-
entiated leaves (Bräutigam et al. 2011a). In addition,
hundreds of proteins have been identified that differ-
entially accumulate in developing maize M cells or
BS cells respectively (Majeran and Van Wijk 2009;
Majeran et al. 2008). Li et al. (2010) performed
RNA-seq analysis of the maize leaf transcriptome
along a leaf developmental gradient to explore the
developmental dynamics of C4 cell-type differentia-
tion. Comparative proteomics of differentiated BS
and M cells of maize demonstrated qualitative and
quantitative differences between M and BS chloro-
plasts (Friso et al. 2010).
The 1KP Project (http://www.onekp.com/) is launched
in November 2008 with the aim to acquire gene se-
quence information of 1 000 plant species using
RNA-Seq technology. Around 100 plant species re-
lated to C3/C4 study are included in this project.
Now, the data of 80 Flaveria transcriptomes are un-
der analysis. Since RNA-seq and proteomics analyses
are available commercially, much more datasets are
generating. Thus, a systems biology approach is re-
quired to analyze these datasets to identify candidate
regulators controlling C4 differentiation (Zhu et al.
2010b). These will generate important new candidate
genes to follow up using reverse genetics approach.
5 Setaria viridis: new model system for C4 photo-
synthesis
As mentioned above, a large-screening for gain of C4
function in rice and loss of C4 function in sorghum
and maize was launched recently by IRRI (Interna-
tional Rice Research Institute, Philippines), together
with CSIRO (Commonwealth Scientific and Industri-
al Research Organization, Australia) and Washington
State University (USA) (Furbank et al. 2009). How-
ever, both maize and sorghum have big stature, re-
quire substantial growth space, several months to set
and mature seeds, and are not easy to manipulate in
the lab. Therefore, these large-scale genetic screens
are laborious, time-consuming, and impractical when
based on the CO2 compensation point in a CO2-con-
trolled chamber. In addition, they all lack efficient
植物生理学报1134
transformation systems. A more suitable model sys-
tem is needed to perform high-throughput genetic
screens.
Brutnell et al. (2010) proposed a new C4 model
species Setaria viridis to facilitate the study of C4
photosynthesis. Setaria viridis (common name: green
foxtail) belongs to the Panicoideae subfamily which
includes important C4 cereals (maize, sorghum, pearl
millet, foxtail millet), major biofuel (sugarcane) and
bioenergy feedstocks Miscanthus and switchgrass. S.
viridis is an NADP-ME subtype C4 grass which uses
an NADP-malic enzyme as decarboxylating enzyme
in bundle sheath cells and is closely related to the
grain crop foxtail millet (Setaria italic).
Setaria viridis is a true diploid with a relatively
small genome of ~510 Mb and has also been se-
quenced (Brutnell et al. 2010). S. viridis meets all the
requirements to be an ideal model system for forward
and reverse genetics, including small size (10–15 cm),
simple growth requirements, a short life cycle (6–9
weeks depending on photoperiod conditions), and
prolific seed production (~13 000 seeds per plant)
(Brutnell et al. 2010; Li and Brutnell 2011). Com-
pared to other C4 plants, another attractive feature of
S. viridis as a genetic system is that Setaria viridis
now has a working transformation system. Brutnell et
al. (2010) successfully regenerated plants from seed
callus, established a transient transformation system,
and developed stable transformation using Agrobacte-
rium tumefaciens–mediated transformation.
Now several international labs including Hui
Zhang’s lab (Shandong Normal University) and our
lab are working on large-scale forward-genetic
screens in mutagenized S. viridis populations for mu-
tants involved in C4 photosynthetic pathway. These
forward genetic screens in Setaria may improve our
understanding C4 photosynthesis as they have done
in Arabidopsis thaliana research.
In summary, not much progress has been made in
defining the regulatory networks for C4 differentia-
tion since C4 photosynthesis was discovered (1960s).
Part of the reason is that few large-scale genetic
screens were done for loss or gain of C4 function.
The technological breakthroughs, especially NGS,
will accelerate our understanding C4 photosynthesis.
New model system, Setaria viridis, provides us un-
precedented genetic manipulation platform for high-
throughput screening and gene expression. It is be-
lieved the C4 rice is feasible and C4 progress will
speed up. However, this engineering effort is by no
means an easy task, its final success will undoubtedly
require a close collaboration among scientists in dif-
ferent disciplines and also sustained funding from
different funding bodies around the world.
References
Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S,
Miyao M, Datta SK (2007). Enhanced photosynthesis rate in
genetically engineered indica rice expressing pepc gene cloned
from maize. Plant Sci, 172: 1204~1209
Bansal KC, Viret JF, Haley J, Khan BM, Schantz R, Bogorad L (1992).
Transient expression from Cab-M1 and Rbcs-M3 promoter
sequences is different in mesophyll and bundle sheath-cells in
maize leaves. Prog Natl Acad Sci USA, 89: 3654~3658
Bauwe H (2011). Photorespiration - The bridge to C4 photosynthesis.
In: Raghavendra AS, Sage RF (eds). C4 Photosynthesis and
Related CO2 Concentrating Mechanisms, Heidelberg-Berlin:
Springer Verlag, 81~108
Björkman O, Pearcy RW, Nobs MA (1970). Hybrids between Atriplex
species with and without B-carboxylation photosynthesis. Photo-
synthetic characteristics. Carnegie Inst Wash Yearb, 69: 640~648
Björkman O, Gauhl E, Nobs MA (1969). Comparative studies of Atri-
plex species with and without B-carboxylation photosynthesis
and their first-generation hybrid. Carnegie Inst Wash Yearb, 68:
620~633
Björkman O, Nobs MA, Berry JA (1971). Further studies on hybrids
between C3 and C4 species of Atriplex. Carnegie Inst Wash
Yearb, 70: 507~511
Bolton JK, Brown RH (1980). Photosynthesis of grass species differ-
ing in carbon dioxide fixation pathways. 5. Response of Panicum
Maximum, Panicum Milioides, and tall fescue (Festuca Arundi-
nacea) to nitrogen nutrition. Plant Physiol, 66: 97~100
Bouton JH, Brown RH, Evans PT, Jernstedt JA (1986). Photosyn-
thesis, leaf anatomy, and morphology of progeny from hybrids
between C3 and C3/C4 Panicum species. Plant Physiol, 80:
487~492
Boynton JE, Nobs MA, Björkman O, Pearcy RW (1970). Hybrids
between Atriplex species with and without B-carboxylation pho-
李圆圆等: C4水稻，我们的新挑战 1135
tosynthesis. Leaf anatomy and ultrastructure. Carnegie Inst Wash
Yearb, 69: 629~632
Bräutigam A, Kajala K, Wullenweber J, Sommer M, Gagneul D, We-
ber KL, Carr KM, Gowik U, Maß J, Lercher MJ, et al. (2011a).
An mRNA blueprint for C4 photosynthesis derived from com-
parative transcriptomics of closely related C3 and C4 species.
Plant Physiol, 155: 142~156
Bräutigam A, Mullick T, Schliesky S, Weber APM (2011b). Critical
assessment of assembly strategies for non-model species mRNA-
Seq data and application of next-generation sequencing to the
comparison of C3 and C4 species. J Exp Bot, 62: 3093~3102
Brown RH, Bouton JH, Evans PT, Malter HE, Rigsby LL (1985).
Photosynthesis, morphology, leaf anatomy, and cytogenetics of
hybrids between C3 and C3/C4 Panicum species. Plant Physiol,
77: 653~658
Brown RH, Bassett CL, Cameron RG, Evans PT, Bouton JH, Black
CC, Sternberg LO, Deniro MJ (1986). Photosynthesis of F1 hy-
brids between C4 and C3-C4 species of Flaveria. Plant Physiol,
82: 211~217
Brown RH, Bouton JH (1993). Physiology and genetics of interspecif-
ic hybrids between photosynthetic types. Annu Rev Plant Phys,
44: 435~456
Brutnell TP, Sawers RJH, Mant A, Langdale JA (1999). BUNDLE
SHEATH DEFECTIVE2, a novel protein required for post-
translational regulation of the rbcL gene of maize. Plant Cell, 11:
849~864
Brutnell TP, Wang L, Swartwood K, Goldschmidt A, Jackson D, Zhu
XG, Kellogg E, Van Eck J (2010). Setaria viridis: a model for
C4 photosynthesis. Plant Cell, 22: 2537~2544
Calsa T, Figueira A (2007). Serial analysis of gene expression in
sugarcane (Saccharum spp.) leaves revealed alternative C4 me-
tabolism and putative antisense transcripts. Plant Mol Biol, 63:
745~762
Cameron RG, Bassett CL, Bouton JH, Brown RH (1989). Transfer of
C4 photosynthetic characters through hybridization of Flaveria
species. Plant Physiol, 90: 1538~1545
Chapman KSR, Hatch MD (1981). Aspartate decarboxylation in
bundle sheath cells of Zea mays and its possible contribution to
C4 photosynthesis. Aust J Plant Physiol, 8: 237~248
Fitter DW, Martin DJ, Copley MJ, Scotland RW, Langdale JA (2002).
GLK gene pairs regulate chloroplast development in diverse
plant species. Plant J, 31: 713~727
Friso G, Majeran W, Huang M, Sun Q, van Wijk KJ (2010). Recon-
struction of metabolic pathways, protein expression, and homeo-
stasis machineries across maize bundle sheath and mesophyll
chloroplasts: large-scale quantitative proteomics using the first
maize genome assembly. Plant Physiol, 152: 1219~1250
Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H, Ono K,
Lee BH, Hirose S, Toki S, Ku MSB, et al (2001). Significant ac-
cumulation of C4-specific pyruvate,orthophosphate dikinase in a
C3 plant, rice. Plant Physiol, 127: 1136~1146
Furbank RT, von Caemmerer S, Sheehy J, Edwards G (2009). C4 rice:
a challenge for plant phenomics. Funct Plant Biol, 36: 845~856
Furbank RT (2011). Evolution of the C4 photosynthetic mechanism:
are there really three C4 acid decarboxylation types? J Exp Bot,
62: 3103~3108
Furumoto T, Hata S, Izui K (1999). cDNA cloning and characteriza-
tion of maize phosphoenolpyruvate carboxykinase, a bundle
sheath cell-specific enzyme. Plant Mol Biol, 41: 301~311
Furumoto T, Hata S, Izui K (2000). Isolation and characterization of
cDNAs for differentially accumulated transcripts between meso-
phyll cells and bundle sheath strands of maize leaves. Plant Cell
Physiol, 41: 1200~1209
Hall LN, Rossini L, Cribb L, Langdale JA (1998). GOLDEN 2: A
novel transcriptional regulator of cellular differentiation in the
maize leaf. Plant Cell, 10: 925~936
Hatch MD (1971). C4 pathway of photosynthesis. Evidence for an
intermediate pool of carbon dioxide and identity of donor C4-
dicarboxylic acid. Biochem J, 125: 425-432.
Hatch MD (1987). C4 photosynthesis: A unique blend of modified
biochemistry, anatomy and ultrastructure. Biophys Acta, 895:
81~106
Hibberd JM, Sheehy JE, Langdale JA (2008). Using C4 photosynthe-
sis to increase the yield of rice - rationale and feasibility. Curr
Opin Plant Biol, 11: 228~231
Hibberd JM, Covshoff S (2010). The regulation of gene expression re-
quired for C4 photosynthesis. Annu Rev Plant Biol, 61: 181~207
Holaday AS, Talkmitt S, Doohan ME (1985). Anatomical and enzy-
mic studies of leaves of a C3xC4 Flaveria F1 hybrid exhibiting
reduced photorespiration. Plant Sci, 41: 31~39
Holaday AS, Brown RH, Bartlett JM, Sandlin EA, Jackson RC (1988).
Enzymic and photosynthetic characteristics of reciprocal F1 hy-
brids of Flaveria Pringlei (C3) and Flaveria Brownii (C4-Like
Species). Plant Physiol, 87: 484~490
Hudspeth RL, Grula JW, Dai Z, Edwards GE, Ku MSB (1992). Ex-
pression of maize phosphoenolpyruvate carboxylase in trans-
genic tobacco - effects on biochemistry and physiology. Plant
Physiol, 98: 458~464
Huxman TE, Monson RK (2003). Stomatal responses of C3, C3-C4
and C4 Flaveria species to light and intercellular CO2 concentra-
tion: implications for the evolution of stomatal behaviour. Plant
Cell Environ, 26: 313~322
Ku MSB, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K,
Hirose S, Toki S, Miyao M, Matsuoka M (1999). High-level
expression of maize phosphoenolpyruvate carboxylase in trans-
genic rice plants. Nat Biotechnol, 17: 76~80
Langdale JA, Kidner CA (1994). Bundle-sheath defective, a mutation
that disrupts cellular-differentiation in maize leaves. Develop-
ment, 120: 673~681
Li MR (1993). Leaf photosynthetic nitrogen-use efficiency of C3 and
C4 cyperus species. Photosynthetica, 29: 117~130
Li P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, Kebrom TH,
Provart N, Patel R, Myers CR, et al (2010). The developmen-
tal dynamics of the maize leaf transcriptome. Nat Genet, 42:
1060~1067
Li P, Brutnell TP (2011). Setaria viridis and Setaria italica, model ge-
netic systems for the Panicoid grasses. J Exp Bot, 62: 3031~3037
Majeran W, Zybailov B, Ytterberg AJ, Dunsmore J, Sun Q, van Wijk
植物生理学报1136
KJ (2008). Consequences of C4 differentiation for chloroplast
membrane Proteomes in maize mesophyll and bundle sheath
cells. Mol Cell Proteomics, 7: 1609~1638
Majeran W, van Wijk KJ (2009). Cell-type-specific differentiation of
chloroplasts in C4 plants. Trends Plant Sci, 14: 100~109
Matsuoka M, Tada Y, Fujimura T, Kanomurakami Y (1993). Tissue-
specific light-regulated expression directed by the promoter of a
C4 gene, maize pyruvate, orthophosphate dikinase, in a C3 plant,
rice. Prog Natl Acad Sci USA, 90: 9586~9590
Matsuoka M, Kyozuka J, Shimamoto K, Kano-Murakami Y (1994).
The promoters of two carboxylases in a C4 plant (maize) direct
cell-specific, light-regulated expression in a C3 plant (rice). Plant
J, 6: 311~319
Matsuoka M, Furbank RT, Fukayama H, Miyao M (2001). Molecular
engineering of C4 photosynthesis. Annu Rev Plant Phys, 52:
297~314
Meister M, Agostino A, Hatch MD (1996). The roles of malate and
aspartate in C4 photosynthetic metabolism of Flaveria bidentis
(L). Planta, 199: 262~269
Morison JIL, Gifford RM (1983). Stomatal sensitivity to carbon-diox-
ide and humidity - a comparison of 2 C3 and 2 C4 grass species.
Plant Physiol, 71: 789~796
Nobs MA, Björkman O, Pearcy RW (1970). Hybrids between Atriplex
species with and without B-carboxylation photosynthesis. Cyto-
genetic and morphological characteristics. Carnegie Inst Wash
Yearb, 69: 625~629
Nomura M, Sentoku N, Nishimura A, Lin JH, Honda C, Taniguchi M,
Ishida Y, Ohta S, Komari T, Miyao-Tokutomi M, et al (2000).
The evolution of C4 plants: acquisition of cis-regulatory se-
quences in the promoter of C4-type pyruvate, orthophosphate
dikinase gene. Plant J, 22: 211~221
Nomura M, Higuchi T, Ishida Y, Ohta S, Komari T, Imaizumi N,
Miyao-Tokutomi M, Matsuoka M, Tajima S (2005a). Differential
expression pattern of C4 bundle sheath expression genes in rice,
a C3 plant. Plant Cell Physiol, 46: 754~761
Nomura M, Higuchi T, Katayama K, Taniguchi M, Miyao-Tokutomi
M, Matsuoka M, Tajima S (2005b). The promoter for C4-type
mitochondrial aspartate aminotransferase does not direct bundle
sheath-specific expression in transgenic rice plants. Plant Cell
Physiol, 46: 743~753
Oguro H, Hinata K, Samejima M, Sugiyama T, Tsunoda S (1988).
Photosynthetic characteristics of the reciprocal interspecific hy-
brids and their progenies between Atriplex Triangularis (C3) and
Atriplex Rosea (C4). Jpn J Breed, 38: 53~64
Pearcy RW, Björkman O (1970). Hybrids between Atriplex species
with and without B-carboxylation photosynthesis. Biochemical
characteristics. Carnegie Inst Wash Yearb, 69: 632~640
Peterhansel C (2011). Best practice procedures for the establishment
of a C4 cycle in transgenic C3 plants. J Exp Bot, 62: 3011~3019
Purcell M, Mabrouk YM, Bogorad L (1995). Red, far-red and blue
light-responsive regions of maize Rbcs-M3 are active in bundle-
sheath and mesophyll-cells, respectively. Prog Natl Acad Sci
USA, 92: 11504~11508
Rossini L, Cribb L, Martin DJ, Langdale JA (2001). The maize
Golden2 gene defines a novel class of transcriptional regulators
in plants. Plant Cell, 13: 1231~1244
Roth R, Hall LN, Brutnell TP, Langdale JA (1996). bundle sheath de-
fective2, a mutation that disrupts the coordinated development of
bundle sheath and mesophyll cells in the maize leaf. Plant Cell, 8:
915~927
Sage RF, Pearcy RW (1987). The nitrogen use efficiency of C3 and
C4 plants. 2. Leaf nitrogen effects on the gas-exchange charac-
teristics of Chenopodium Album (L) and Amaranthus Retroflexus
(L). Plant Physiol, 84: 959~963
Sage RF, Christin PA, Edwards EJ (2011a). The C4 plant lineages of
planet Earth. J Exp Bot, 62: 3155~3169
Sage RF, Zhu XG (2011b). Exploiting the engine of C4 photosynthe-
sis. J Exp Bot, 62: 2989~3000
Schmitt MR, Edwards GE (1981). Photosynthetic capacity and ni-
trogen use efficiency of maize, wheat, and rice - a comparison
between C3 and C4 photosynthesis. J Exp Bot, 32: 459~466
Viret JF, Mabrouk Y, Bogorad L (1994). Transcriptional photoregula-
tion of cell-type-preferred expression of maize Rbcs-M3 - 3
and 5 sequences are involved. Prog Natl Acad Sci USA, 91:
8577~8581
Wang Z, Gerstein M, Snyder M (2009). RNA-Seq: a revolutionary
tool for transcriptomics. Nat Rev Genet, 10: 57~63
Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, Langdale
JA (2009). GLK Transcription factors coordinate expression
of the photosynthetic apparatus in Arabidopsis. Plant Cell, 21:
1109~1128
Westhoff P, Engelmann S, Wiludda C, Burscheidt J, Gowik U, Schlue
U, Koczor M, Streubel M, Cossu R, Bauwe H (2008). The gene
for the P-subunit of glycine decarboxylase from the C4 spe-
cies Flaveria trinervia: Analysis of transcriptional control in
transgenic Flaveria bidentis (C4) and Arabidopsis (C3). Plant
Physiol, 146: 1773~1785
Wingler A, Walker RP, Chen ZH, Leegood RC (1999). Phospho-
enolpyruvate carboxykinase is involved in the decarboxylation
of aspartate in the bundle sheath of maize. Plant Physiol, 120:
539~545
Wostrikoff K, Stern D (2007). Rubisco large-subunit translation is
autoregulated in response to its assembly state in tobacco chloro-
plasts. Prog Natl Acad Sci USA, 104: 6466~6471
Zhu XG, Long SP, Ort DR (2008). What is the maximum efficiency
with which photosynthesis can convert solar energy into bio-
mass? Curr Opin Biotechnol, 19: 153~159
Zhu XG, Long SP, Ort DR (2010a). Improving Photosynthetic Effi-
ciency for Greater Yield. Annu Rev Plant Biol, 61: 235~261
Zhu XG, Shan L, Wang Y, Quick WP (2010b). C4 rice - an ideal
arena for systems biology research. J Integr Plant Biol, 52:
762~770