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Strategies for engineering a two-celled C4 photosynthetic pathway into rice

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DOI: 10.1093/jxb/err022 · Source: PubMed

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 Journal of Experimental Botany Advance Access published February 18, 2011

Journal of Experimental Botany, Page 1 of 10

doi:10.1093/jxb/err022

REVIEW PAPER

Strategies for engineering a two-celled C4 photosynthetic

pathway into rice
Kaisa Kajala1, Sarah Covshoff1, Shanta Karki2, Helen Woodfield1, Ben J. Tolley1, Mary Jaqueline A. Dionora2,
Reychelle T. Mogul2, Abigail E. Mabilangan2, Florence R. Danila2, Julian M. Hibberd1 and William P. Quick2,*
1
Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge CB2 3EA, Cambridge, UK
2
International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines

Downloaded from jxb.oxfordjournals.org at National Taiwan University on February 22, 2011

* To whom correspondence should be addressed: E-mail: w.p.quick@irri.org

Received 8 November 2010; Revised 13 January 2010; Accepted 19 January 2011

Abstract
Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with
a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future,
rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C4 photosynthetic
cycle into rice. The international C4 Rice Consortium was founded in order to test the feasibility of installing the C4
engine into rice. This review provides an update on two of the many approaches employed by the C4 Rice
Consortium: namely, metabolic C4 engineering and identification of determinants of leaf anatomy by mutant screens.
The aim of the metabolic C4 engineering approach is to generate a two-celled C4 shuttle in rice by expressing the
classical enzymes of the NADP-ME C4 cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and
glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C4-like fashion by specifically
down-regulating their expression in rice mesophyll (M) cells. In addition to the changes in biochemistry, two-celled
C4 species show a convergence in leaf anatomy that include increased vein density and reduced numbers of M cells
between veins. By screening rice activation-tagged lines and loss-of-function sorghum mutants we endeavour to
identify genes controlling these key traits.

Key words: Activation-tagged lines, C4 engineering, C4 photosynthesis, maize, metabolic engineering, mutant screens, rice,
transformation.

Introduction
Rice provides the majority of the calorific intake for the
world’s population. If we are to provide food for the
predicted world population of 9 billion people in 2050 we
need to generate substantial increases in the yield of the rice
crop. Currently, it is estimated that 925 million people
suffer from chronic hunger and about 14 400 children die of
hunger-related causes every day (FAO, 2010). Population
growth in Asia will require a 60% increase in rice pro-
duction and so each rice-producing hectare that currently
feeds 27 people will need to provide food for 43 people in
future (Sheehy et al., 2008). Additional constraints will
be put on farming and land usage by climate change,
decreasing water and energy availability and the demand
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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for biofuels will further exacerbate constraints on food
production (Rosegrant et al., 2002; Tilman et al., 2009).
Increases in crop yields have matched population growth
until recently (Mitchell and Sheehy, 2006), but the high
yields associated with breeding for short-stature cereal
cultivars appear to have reached a plateau (Kropff et al.,
1994). A second Green Revolution has been called for in
order to accelerate the increase in crop yields that will be
required to support the projected population growth.
Current breeding programmes will contribute to increasing
crop yields (Tester and Langridge, 2010) and will also allow
us to utilize marginal land area by breeding for drought and
salt-tolerant cultivars (Gregorio et al., 2002; Flowers, 2004).
2 of 10 | Kajala et al.
However, these approaches are unlikely to be sufficient to
increase rice yield by the 60% required in 2050.
Modelling and empirical analysis have shown that
maximum crop yield is determined by the amount of light
incident on a crop over the course of its growing season, the
proportion of light intercepted by the crop, the photosyn-
thetically active radiation use efficiency (RUE), and the
harvest index (Mitchell and Sheehy, 2006; Long et al.,
2006). The harvest index and the fraction of photosynthet-
ically active radiation (PAR) intercepted were significantly
increased during the Green Revolution. While yields can be
increased by extending the duration of growth, per unit time
this does not increase the amount of dry matter harvested.
The amount of PAR incident on a crop can only be
modified by growing that crop in sunnier climes. Sub-
sequent to the Green Revolution, modifications to RUE are
likely to achieve the most significant increases in crop yield.
Fortunately, the evolution of C4 photosynthesis provides
a biological precedent that implies RUE of C3 crops can be
increased. C4 species such as maize, sorghum, and sugar-
cane have 50% higher RUEs than those of C3 crops such as
rice, wheat, and potato (Kiniry et al., 1989; Sage, 2004).
Introducing the C4 photosynthetic cycle into rice could
allow a 50% increase in the RUE and crop yield (Mitchell
and Sheehy, 2006; Hibberd et al., 2008), as well as
potentially improve nitrogen and water use efficiencies
(Sage and Pearcy, 1987; Makino et al., 2003; Sage, 2004).
In order to test the feasibility of introducing a two-celled
C4 cycle into rice, an international C4 Rice Consortium
led by the International Rice Research Institute (IRRI,
Philippines) with 24 participating research groups was set
up in 2008 (http://irri.org/c4rice). Within this Consortium,
activities range from systems biology, physiological pheno-
typing, and deep sequencing, through to metabolic engi-
neering and screening of rice mutants for characteristics of
Kranz anatomy. Overall, these complementary approaches
should increase our understanding of the C4 pathway and
facilitate attempts to engineer it into C3 species. It is clear
that alternative approaches such as modifying existing
(Lefebvre et al., 2005) and engineering new metabolic
pathways (Kebeish et al., 2007; Bar-Even et al. 2010) could
also lead to increases in RUE. In this article, an update is
provided on the approaches being taken towards metabolic
engineering and identifying lines of rice and sorghum
mutants with modification to vein spacing. To place this
work into context, a brief summary of how the C4 pathway
operates in most terrestrial C4 plants will be provided and
then the strategies being used to test the feasibility of
placing the C4 pathway into rice will be explained.
The central enzyme of the Calvin–Benson cycle, ribulose
bisphosphate carboxylase oxygenase (RuBisCO), catalyses
not only the fixation of CO2 but also of O2. The oxygenase
activity of RuBisCO produces a toxic metabolite, phospho-
glycolate (Bowes et al., 1971), which is then broken down
into an energy-consuming series of reactions known as
photorespiration (Tolbert, 1971). The ratio of carboxylation
to oxygenation reactions depends on the ratio of soluble
CO2 and O2 present, and this ratio decreases with increasing
temperature. Hence, C3 photosynthesis becomes more
costly and its RUE decreases at higher temperatures.
The C4 photosynthetic cycle supercharges photosynthesis
by concentrating CO2 around RuBisCO and significantly
reduces the oxygenase reaction. Although the C4 pathway
incurs an additional cost of 2 ATP per CO2 fixed, it is less
energy-demanding than C3 photosynthesis at leaf temper-
atures over 20–25
C due to the temperature-dependence of
the oxygenation reactions (Ehleringer and Björkman, 1977;
Ehleringer and Pearcy, 1983), making C4 species more
productive in environments where rice is farmed. The C4
cycle found in species that have been domesticated into
high-yielding crops employ the two-celled C4 system, with
mesophyll (M) and bundle sheath (BS) cells positioned in
concentric rings around the vascular tissue in so-called
Kranz anatomy (Dengler and Nelson, 1999). The biochem-
Downloaded from jxb.oxfordjournals.org at National Taiwan University on February 22, 2011
ical reactions of the C4 cycle are spatially separated between
these two cell types (Hatch, 1971) and the expression of C4
genes encoding these proteins are regulated in M or
BS-specific fashions (Sheen, 1999; Hibberd and Covshoff,
2010). The first committed step of the C4 cycle occurs in
M cells where initial carbon fixation is catalysed by
phosphoenolpyruvate carboxylase (PEPC) forming the
four-carbon compound oxaloacetate (OAA) from bicarbon-
ate and PEP. OAA is then metabolized into either malate or
aspartate depending on the biochemical subtype (Furbank,
2011), and the four-carbon acid then diffuses into the BS
cells where it is decarboxylated to provide increased
concentrations of CO2 in BS cells within which RuBisCO is
confined. Finally, the initial substrate of the C4 cycle, PEP,
is regenerated in M cells by pyruvate, orthophosphate
dikinase (PPDK). In addition to these alterations to the
biochemistry of photosynthesis and those to leaf develop-
ment, C4 leaves also have modified cell biology such as
changes to the organelle structure. Considering all these
layers of complexity, the independent evolution of C4
photosynthesis at least 62 times in 19 different families of
angiosperms (Sage et al., 2011) indicates that it is one of the
most remarkable examples of convergent evolution.
Engineering the C4 cycle
There is no doubt that introducing a multigenic trait such as
C4 photosynthesis into rice is an enormous challenge.
However, one compelling reason to accept this challenge is
the strong humanitarian case for significantly increasing
RUE. In addition, there are a number of biological reasons
that indicate that the evolution of C4 photosynthesis may
not be as difficult as first appears. First, C4 photosynthesis
has evolved multiple times indicating that C3 species may in
some ways be preconditioned for the C4 pathway (Sage,
2004). Second, to our knowledge, no new genes are
associated with the C4 pathway: all enzymes of the C4 cycle
are found in C3 species although they often accumulate to
low levels and are thought to fulfill housekeeping functions.
In fact, C3 plants possess characteristics of the C4 pathway
in cells close to the veins of stems and petioles (Hibberd and
Quick, 2002; Brown et al., 2010). Third, although there is

only one study to date, the current estimate is that
approximately 3% of the mature leaf transcriptome differs
between closely related C3 and C4 species (Bräutigam et al.,
2010). Fourth, it is possible that a good proportion of these
changes to the leaf transcriptome are due either to
secondary effects associated with the alterations to primary
metabolism in a C4 leaf or other processes associated with
phylogenetic distance between these species. This can be
tested to some extent by assessing the impact of partitioning
a significant portion of the C4 cycle into M and BS cells of
rice. Lastly, recent advances in sequencing technologies,
proteomics, and the availability of multiple C4 genomes
(Paterson et al., 2009; Schnable et al., 2009; Li et al., 2010)
has opened the C4 pathway to experimental dissection using
a systems biology approach. Comparative transcriptomics
and proteomics have provided insights into the partitioning
of C4 gene expression and protein accumulation through
analysis of M and BS cells in maize (Majeran et al., 2005,
2008; Sawers et al., 2007; Covshoff et al., 2008; Friso
et al., 2010), between M and BS cells of rice (Jiao et al.,
2009), along the maize leaf developmental gradient (Li
et al., 2010), and between both distantly related (Bräutigam
et al., 2008) and closely related C3 and C4 species
(Bräutigam et al., 2010). In future, the systems biology
approach will be extended to multiple lineages of closely
related C3 and C4 species to reveal core elements of C4
biology. Considering all these points it seems sensible to
start to attempt to engineer the two-celled C4 cycle into rice.
Factors influencing the current strategy to
build a two-celled C4 cycle in rice leaves
The approaches used to test the feasibility of compartment-
ing the C4 cycle into M and BS cells of rice are based on
knowledge of previous studies combined with the following
points.
(i) The promoters of several genes from C4 species have
been characterized in rice, and this has provided tools to
enable cell type-specific expression in rice. For example,
the ZmPEPC and ZmPPDK promoters generate strong
M-specific expression in rice, whereas the ZjPCK1 pro-
moter drives expression in BS and vascular cells of rice
(Matsuoka et al., 1993, 1994; Nomura et al., 2000, 2005).
(ii) Correct regulation of enzyme activities is an important
consideration for C4 engineering. For example, both
PEPC and PPDK are activated in the light in C4 species
via post-translational modification by PEPC kinase and
the PPDK regulatory protein respectively (Nimmo
et al., 1987; Burnell and Chastain, 2006; Chastain et al.,
2008). However, phosphorylation of maize PEPC does
not follow this pattern in rice (Fukayama et al., 2003).
The incorrect regulation or constitutive activity of C4
enzymes will complicate C4 cycle function and could
generate futile pathways (Taniguchi et al., 2008).
(iii) The C4 cycle requires extensive transport of metabolites
between different subcellular compartments, but cur-
Two-celled C4 photosynthesis in rice | 3 of 10
rently there are limited numbers of candidate proteins
for these steps (Taniguchi et al., 2004; Bräutigam et al.,
2008; Majeran et al., 2008; Weber and Von Caemmerer,
2010). The transporters are an important component of
C4 engineering, as the cycle likely cannot function
efficiently without increased fluxes of C4 metabolites
across organellar membranes (Bräutigam et al., 2008;
Weber and Von Caemmerer, 2010).
(iv) Rice contains a chloroplastic PEPC that has the
potential to create futile cycling of the metabolites in
the chloroplasts of rice (Masumoto et al., 2010). It is
proposed that the chloroplast localization of PEPC in
rice is important for nitrogen assimilation in anaerobic
conditions. This finding may complicate attempts to
place a C4 system into rice. One way of addressing this
issue is to determine how the C4 cycle works in the
Downloaded from jxb.oxfordjournals.org at National Taiwan University on February 22, 2011
aquatic C4 plant Echinochloa spp. that grows alongside
rice as a weed in paddies.
(v) Attempts at generating a single-celled C4 system have
not yet led to a functional C4 cycle in leaves of C3
species. It has been recognized for many years that the
C4 pathway is associated with increased yields, so there
have already been significant efforts aimed at engineer-
ing genes of C4 metabolism into various C3 species.
This has included introducing a partial C4 cycle into
the mesophyll cells of rice, potato, and tobacco. These
studies have been reviewed extensively (Matsuoka et al.
2001; Häusler et al., 2002; Leegood, 2002; Miyao,
2003; Raines, 2006; Burnell, 2007; Taniguchi et al.,
2008; Miyao et al., 2011). Over-expression of specific
genes in rice as well as increases in the enzyme activities
that they encode have been achieved (Miyao, 2003).
However, negative impacts on growth were not un-
common (Raines, 2006). More recently, rice trans-
formants have been generated with a set of genes
(PEPC, MDH, NADP-ME, and PPDK) that would
allow a basic single cell C4-like biochemical cycle to
operate (Taniguchi et al., 2008; Miyao et al, 2011).
These transgenic plants were often stunted, possibly
due to the generation of futile cycles and a lack
of correct circadian regulation of enzyme activity
(Taniguchi et al., 2008). It should be noted that the
expression of the transporters necessary for organelle
metabolite exchange have, to date, not been modified
in these plants. It is also noteworthy that plants with
the native ability to concentrate CO2 within a single
cell (Reiskind et al., 1989; Voznesenskaya et al., 2001)
appear to rely on the evolution of very large cells
within which two populations of chloroplasts are
separated by large distances in order to minimize the
diffusion of CO2 away from its release around
RuBisCO (Edwards et al., 2004). In addition, the most
productive C4 crops use the two-celled system. There-
fore, while continued work on single-celled species may
help our understanding of C4 photosynthesis, it would
therefore appear sensible to assess the feasibility of
generating a two-celled C4 system in C3 leaves.
4 of 10 | Kajala et al.
Introducing the genes of C4 photosynthesis
into rice
While many projects are underway within the C4 Rice
Consortium to achieve this goal, our focus has been on
generating resources that will allow us to test the feasibility
of generating a functional biochemical C4 pathway in rice.
The NADP-ME subtype was chosen for C4 engineering
(Fig. 1) as it is well-characterized in the C4 crop and model
species maize, and, of the three biochemical sub-types, it
potentially requires the fewest enzymes and transporters for
the C4 shuttle (Weber and Von Caemmerer, 2010). In order
to engineer a functional C4 shuttle into rice, genes are being
cloned from maize and introduced individually into rice. In
addition, endogenous rice genes are being down-regulated.
The first stage of the project is to integrate the classical
genes encoding the C4 metabolic enzymes (Fig. 1) into rice
and to assess the extent to which they lead to high levels of
expression in the correct cell type. The functional two-cell
Fig. 1. Summary of NADP-ME subtype biochemistry and the first
stage of C4 engineering into rice. Initial carboxylation occurs in
mesophyll (M) cells via PEP carboxylase (PEPC) catalysing the
formation of oxaloacetate (OAA) from bicarbonate and phospho
enolpyruvate (PEP). OAA is converted into malate in M chloroplasts
by malate dehydrogenase (MDH) and then malate diffuses into
bundle sheath (BS) cells via plasmodesmata. Decarboxylation
occurs in BS chloroplasts via NADP-malic enzyme (NADP-ME),
increasing the CO2 concentration in the BS to 1–2%. RuBisCO is
expressed in a BS-specific fashion and the increased CO2
concentration suppresses its oxygenation activity. The decarboxy-
lation product, pyruvate, is regenerated into the primary carbon
acceptor PEP in the M chloroplasts by pyruvate, orthophosphate
dikinase (PPDK) in an ATP-dependent fashion. The second
substrate for the cycle, bicarbonate, is formed from CO2 by
cytosolic carbonic anhydrase (CA) in M cells. The dark circles
represent transporters important for C4 metabolite fluxes into and
out of organelles and the dashed line represents cell wall and the
plasmodesmata. The first stage of engineering the two-celled C4
cycle into rice involves expression of CA, PEPC, MDH, and PPDK
in M cells and NADP-ME in BS cells. Also, C4-like down-regulation
of RuBisCO and glycine decarboxylase (GDC, not shown) in M
cells is being carried out with artificial microRNAs against RuBisCO
small subunit (RbcS) and GDC H-subunit (GDC-H) genes.
C4 cycle also requires down-regulation of part of the
Calvin–Benson cycle in M cells. We are testing whether
artificial microRNAs (amiRNAs) driven by cell specific
promoters can be used to down-regulate processes specifi-
cally in the M. Artificial microRNAs (amiRNAs) against
the rice RuBisCO small subunit (RbcS) genes under the
control of an M-specific promoter have been engineered.
One of the initial steps in the evolution of the C4 pathway is
proposed to be the repositioning of photorespiration into
the BS cells of C3 leaves (Rawsthorne et al., 1988; Morgan
et al., 1993). To test the importance of this modification,
plants have been generated in which amiRNAs that target
glycine decarboxylase subunit H (GDC-H) for degradation
are expressed in M cells. With these plants, the aim was to
test whether BS-specific accumulation of GDC is necessary
to prime a plant for C4 cycle function and whether it also
Downloaded from jxb.oxfordjournals.org at National Taiwan University on February 22, 2011
initiates additional pleiotropic effects that are associated
with a C4 leaf. The second stage of C4 engineering will
include addition of the transporters required to support
increased fluxes of metabolites between subcellular com-
partments of the C4 cycle. Candidate transporters have been
identified from differential proteomic studies (Majeran
et al., 2005, 2008; Bräutigam et al., 2008; Friso et al., 2010)
by high accumulation in C4 relative to C3 leaves as well
as appropriate cell type-specific accumulation in maize.
The transporters are being cloned from maize and include
a putative OAA/malate antiporter (OMT1), putative dicar-
boxylate transporters (DiT1 and DiT2), and the PEP/
phosphate translocator (PPT1). A possible third stage to
the C4 rice project will include the incorporation of
additional genes identified by members of the consortium
as candidates controlling leaf anatomy or cell biology
associated with the C4 leaf.
All of the genes encoding components of the C4 pathway
that are introduced into rice need to be expressed correctly
in either a M or BS-specific fashion (Fig. 1). The M-specific
expression patterns of maize promoters in rice have been
characterized for ZmPEPC and ZmPPDK (Matsuoka et al.,
1993, 1994; Nomura et al., 2000), and these promoters have
been successfully used to drive strong M-specific expression
in rice (Ku et al., 1999; Fukayama et al., 2001; Suzuki et al.,
2006). Although the intact genes for PEPC and PPDK have
also been placed into rice, to our knowledge, they have not
been reported to generate cell specificity. In addition, for
other genes, it is clear that cell specificity in C4 leaves can be
mediated by elements that are not present in the promoter
(Hibberd and Covshoff, 2010). Therefore, in order to
maximize the chances of obtaining correct cell specificity as
well as other types of regulation (e.g. light) for each gene,
the full-length maize gene has been cloned (including the
native promoter, 5# and 3# UTRs, exons, and introns) and
placed into rice. It is possible that some of these genes will
not generate cell specificity (Nomura et al., 2005) and so
each coding sequence (CDS) has also been fused to either
the M-specific ZmPEPC promoter (Matsuoka et al., 1993)
or the BS promoter OsPCK1 (Nomura et al., 2005) with the
nopaline synthase (nos) terminator at the 3# end. This
approach should help to ensure that the correct expression

pattern will be obtained for each gene. An additional
difference between our strategy and previous ones is that
we are attempting to separate our gene of interest from the
presence of a strong proximal promoter. The strong CaMV
35S promoter commonly used to drive expression of plant
selectable markers can have a long-distance enhancing effect
on the expression of the introduced gene of interest (Yoo
et al., 2005), and this has the potential to disrupt cell
specificity of C4 gene expression in rice. The CaMV 35S
promoter and the plant selectable marker from the maize
gene of interest have therefore been separated into two
different plasmids that are then co-transformed into rice
(Komari et al., 1996). Although it is anticipated that co-
integration at the same locus will still occur (Kohli et al.,
1998), plants with unlinked sites of insertion can be selected
and the plant selectable marker can be crossed out when
necessary for future studies.
In order to express the C4 shuttle in rice, the constructs
are being introduced individually into Indica rice IR64.
Lines with low copy numbers and high, stable expression of
each transgene are being identified, and then stacked
together by crossing. Rice transformation is performed by
Agrobacterium-mediated transformation of immature rice
embryos (Hiei and Komari, 2006). After antibiotic selec-
tion and regeneration of resistant calli, transformed T0
plants are produced. As the C4 cycle will be stacked
together by crossing, the optimal transgenic plant would
only have a single insertion of the transgene into the rice
genome. However, the majority of the T0 plants contain
multiple insertions, so low-copy number plants with high
expression levels of the transgene are chosen to produce T1
and T2 generations and are selected later for single copy
inserts by segregation analysis. In order to build a func-
tional C4 cycle, high expression levels of the C4 genes in the
correct cell type are required. Hence, each generation of
transgenic plants is being tested for expression of each
transgene by two approaches: transcript abundance is
determined by quantitative RT-PCR and protein abun-
dance is being assessed by immunoblotting and enzyme
activity assays. To assess whether each protein accumulates
in the correct cell type, a combination of immunolocaliza-
tion and mRNA localization in situ is being used. To
determine whether introducing components of the C4 cycle
into rice leaves has an impact on leaf development or cell
ultrastructure, as has been reported with other manipu-
lations of primary metabolism (Takeuchi et al., 2000;
Pellny et al., 2004; Raines and Paul, 2006), organelle
structure and leaf anatomy are being monitored in each
line. Finally, the transgenic plants with correct localization
and significant activity of each protein will be crossed in
order to start stacking the transgenes to produce a pro-
totype of a rice plant with a fully functional two-cell C4
shuttle.
The prototype of transgenic C4 rice will require detailed
characterization. The biochemistry will be assayed for
correct enzyme function and C4 cycle activity will be
confirmed with carbon fixation studies. Physiological
processes, such as gas exchange and efficiency of photo-
Two-celled C4 photosynthesis in rice | 5 of 10
chemistry, will be analysed for improvements in compen-
sation point and quantum yield expected for C4
photosynthesis. If these results provide cause for opti-
mism, it is likely that emerging technologies such as site-
specific recombination or artificial chromosome engineer-
ing will have to be used to reduce the number of
transgenic events associated with these plants, and these
second generation approaches used to cross traits into
elite lines. Confined field trials would then be conducted
to characterize the effect of the C4 engine on rice yield and
the parameters contributing to it. Subsequently, C4
photosynthesis would be transferred to elite rice varieties
adapted to a wide range of environmental and agronomic
conditions by conventional breeding.
Engineering C4 leaf anatomy
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Although the basic biochemistry of C4 photosynthesis has
been understood for over 40 years, many other aspects of
C4 photosynthesis remain unexplained. To our knowledge,
none of the genes controlling C4 leaf anatomy have been
identified, although the changes in leaf anatomy are well-
characterized and integral to two-celled C4 photosynthesis
(Dengler and Nelson, 1999). Similarly uncharacterized are
the genetic determinants for the majority of C4 cell biology
and ultrastructure, including the genes controlling suberiza-
tion of BS cells, increased plasmodesmatal connectivity
between M and BS cells, and the production of dimorphic
chloroplasts and their intracellular positioning. In order to
engineer these structural C4 changes into rice, the genetic
determinants need to be identified.
The patterns of leaf vascular systems are highly varied in
both C3 and C4 species (Nelson and Dengler, 1997; Dengler
and Kang, 2001; Roth-Nebelsick et al., 2001; McKown and
Dengler, 2007). The leaves of two-cell C4 systems exhibit
increased vein density (Takeda and Fukuyama, 1971;
Dengler et al., 1994, Ueno et al., 2006), which decreases the
diffusion distance of C4 acids from M to BS cells and the
transport distance of the photosynthate into the vasculature
(Hatch, 1987). It has been suggested that the veins of C4
species play a role in determining tissue differentiation and
gene expression (Langdale and Nelson, 1991). Comparisons
of the vein densities of leaves from C3, C3–C4 intermediate,
and C4 Flaveria species suggest that the increased vein
density appeared prior to the evolution of C4 biochemistry
(McKown and Dengler, 2007). A theoretical optimum for
vein density has been suggested (Noblin et al., 2008),
but there is probably adaptive value for variation in vein
density in C3 leaves in response to environmental conditions
(Roth-Nebelsick et al., 2001). For example, C3 leaves grown
under high irradiance can develop denser venation in order
to allow more cooling transpiration (Roth-Nebelsick et al.,
2001; Sage, 2001; Sack and Frole, 2006). In order to
understand the variation in vein density, two genetic screen
approaches were chosen: a screen for emergence of C4
characteristics in activation-tagged rice populations and
a screen for loss of C4 characteristics in C4 sorghum mutant
populations (Fig. 2).
6 of 10 | Kajala et al.

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Fig. 2. Vein spacing and M cell number is reduced in C4 compared with C3 species. Simple anatomical assessments of rice (C3, left
column) and sorghum (C4, right column) mutants are being carried out in order to screen for changes in leaf anatomy. The vein density of
IR64 rice is 5.5 mm 1 (A) whereas in sorghum vein density is 8.0 mm 1 (B). Cross-sections of the leaves are used to count M cell
number between veins. On average rice leaves contain seven (C) and sorghum leaves only two M cells (D) between the veins. In order to
study the emergence and loss of C4-like leaf anatomy, rice activation-tagged lines and sorghum ethyl methanesulphonate (EMS) and
gamma irradiated mutant lines are being screened for changes in these characteristics.

To study the emergence of C4 traits in C3 rice, gain-of-
function genetics is being employed. Activation-tagging
utilizes enhancer elements randomly inserted into the
genome (Hayashi et al., 1992; Weigel et al., 2000), which
creates lines with increased expression or knock out of
specific genes. Activation-tagged rice populations for Indica
and Japonica rice (Jeong et al., 2002, 2006) are being
screened for decreased vein spacing and C4-like leaf
anatomical characteristics. 60 000 plants are being screened,
and vein density is affected in 0.1% of the activation-tagged
plants. Preliminary results are promising, as some of the
mutant lines have increased vein densities and importantly
reduced numbers of M cells between veins. These rice lines
with increased vein density can be directly used as acceptor
lines for the engineered C4 biochemistry. Complementarily,
classical loss-of-function mutant populations of sorghum
were generated to study the loss of C4 characteristics.
Sorghum mutant populations were generated with ethyl
methanesulphonate (EMS) and gamma ray irradiation
treatments, and over 70 000 M2 mutants are currently being
screened. Mutants with decreased vein density and an
increasing number of M cells between veins have been
successfully identified. These potential C4 vein density genes
from both rice and sorghum mutants can be identified and
used for C4 engineering.
The rice and sorghum mutant populations are also
screened for the emergence and loss of physiological C4
characteristics. The carbon concentrating mechanism of C4
plants allow them to carry out net carbon assimilation at
low CO2 concentrations, indicated by a low carbon
compensation point. Hence, sorghum mutants are screened
for growth at low CO2 concentration (20 ppm) where
mutants that exhibit stunted growth are then rescued in
a high-CO2 environment (10 000 ppm). Preliminary results
show the coincidence of increased carbon compensation
point and decreased vein density in the sorghum mutants.
The efficiency of C4 plants also coincides with alterations in
leaf chlorophyll and a/b ratio (Black and Mayne, 1970), and
the activation-tagged rice mutants are screened for changes
in chlorophyll content. Interestingly, breeding and evolution
has led to a reduced carbon compensation point in rice
compared with typical C3 values at high temperatures (Sage
and Sage, 2009). This is believed to be due to a non-C4
photorespiratory compensation mechanism arising from the
lobed shape of M cells, and it may have as yet unknown
implications for C4 rice engineering (Sage and Sage, 2009).
Regardless of the lack of understanding of the genetic
determinants of the leaf anatomy, the mutant screen
approaches are providing promising results for C4-like rice
leaves.
Conclusions
The current rate of increase in crop yields is not sufficient
to feed the increasing world population. C4 species can
have 50% higher yields than C3 species, and in order to
increase its yield potential, engineering C4 photosynthesis
into rice is being attempted by the international C4 Rice
Consortium. The two-celled C4 cycle was chosen for this
project. The direct engineering approach involves express-
ing genes encoding the classical NADP-ME subtype
pathway in appropriate cells in the rice leaf. To replicate
the C4 cycle, RuBisCO and GDC expression are being
down-regulated in M cells. Transgenic plants are currently

being produced and screened for C4 enzyme activity and
localization. To build up the C4 pathway, classical breeding
will be used. Future steps include the addition of C4
transporters as well as uncharacterized genes that are being
discovered by other members of the Consortium. In
a complementary part of the programme, large-scale
mutant screens of sorghum and activation-tagged lines of
rice are being carried out to identify genes controlling vein
density and C4-like characteristics. Combined with other
approaches being undertaken by other members of the
Consortium it is hoped that these approaches will greatly
enhance our understanding of C4 photosynthesis and
further the C4 engineering effort.
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