Journal of Experimental Botany, Vol. 62, No. 9, pp. 3011–3019, 2011
doi:10.1093/jxb/err027 Advance Access publication 18 February, 2011
REVIEW PAPER
Best practice procedures for the establishment of a C4 cycle
in transgenic C3 plants
Christoph Peterhansel*
Institute of Botany, Leibniz University Hannover, Herrenhaeuser Straße 2, D-30419 Hannover, Germany
* To whom correspondence should be addressed. E-mail: [email protected]
Received 1 November 2010; Revised 20 January 2011; Accepted 21 January 2011
Abstract
C4 plants established a mechanism for the concentration of CO2 in the vicinity of ribulose-1,5-bisphosphate
carboxylase/oxygenase in order to saturate the enzyme with substrate and substantially to reduce the alternative
fixation of O2 that results in energy losses. Transfer of the C4 mechanism to C3 plants has been repeatedly tested,
but none of the approaches so far resulted in transgenic plants with enhanced photosynthesis or growth. Instead,
often deleterious effects were observed. A true C4 cycle requires the co-ordinated activity of multiple enzymes in
different cell types and in response to diverse environmental and metabolic stimuli. This review summarizes our
current knowledge about the most appropriate regulatory elements and coding sequences for the establishment of
C4 protein activities in C3 plants. In addition, technological breakthroughs for the efficient transfer of the numerous
genes probably required to transform a C3 plant into a C4 plant will be discussed.
Key words: C4 photosynthesis, gene transfer, promoter, protein modification.
Introduction
The International Rice Research Institute (IRRI) and
associated partners aim to transfer C4 properties to C3 plants
(Hibberd et al., 2008). The anticipated long-term objective is
that future rice varieties will perform true C4 photosynthesis.
This aim is desirable because of the higher yield potential of
C4 compared with C3 plants resulting from the greater
photosynthetic conversion efficiency (Zhu et al., 2008b). Up
to 50% higher possible yields are estimated if rice would
perform C4 photosynthesis and such yield increases will
probably be required by 2050 to feed the ever-growing world
population (Mitchell and Sheehy, 2006). The greater photosynthetic efficiency of C4 plants is based on a biochemical
CO2 pump that saturates the CO2-fixing enzyme ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) with substrate
(reviewed in Kanai and Edwards, 1999). To this end,
Rubisco is confined to bundle sheath cells around veins with
little or no contact with the intercellular air space. Primary
CO2 uptake takes instead place in mesophyll cells that are in
direct contact to BS cells, but also exposed to the air space.
The gas is first hydrated and dissolved to HCO3 by carbonic
anhydrase. HCO3 is then fixed by phosphoenolpyruvate
carboxylase (PEPC) forming oxaloacetate (OAA) from
phosphoenolpyruvate (PEP). The name C4 plant is derived
from the four C atoms of OAA. Dependent on the C4
subtype, aspartate can be alternatively formed and converted
back to malate and/or oxaloacetate in the bundle sheath.
Any of the C4 acids diffuses into the bundle sheath and is
decarboxylated here by one of three decarboxylases that
define the three different subtypes of C4 biochemistry: (i)
NADP-dependent malic enzyme (ME) in the chloroplast, (ii)
NAD-dependent ME in the mitochondrion, or (iii) phosphoenolpyruvate carboxykinase (PCK) in the cytoplasm. In all
cases, CO2 is released that can be efficiently refixed by
Rubisco. The resulting C3 acids diffuse back into the
mesophyll. In the PCK subtype, PEP is directly formed from
malate and available for the next HCO3 fixation, whereas
NADP-ME and NAD-ME plants need to recycle PEP by the
phosphorylation of pyruvate. This reaction again takes place
in the mesophyll chloroplasts and is catalysed by pyruvatePi-dikinase (PPDK). It is a major advantage of this
biochemical pathway compared with conventional C3 photosynthesis that PEPC does not accept oxygen as an alternative
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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3012 | Peterhansel
substrate. This property of Rubisco normally results in the
formation of high amounts of phosphoglycolate that have to
be recycled in the energy-consuming photorespiratory pathway (Maurino and Peterhansel, 2010). C4 plants manage to
enhance CO2 concentration in bundle sheath cells by a factor
of 5 or more relative to mesophyll cell concentrations (Dai
et al., 1993), thus efficiently suppressing the oxygenation of
ribulose-1,5-bisphosphate.
C4 metabolism independently evolved more than 50 times
in various monocot and dicot families (Sage, 2004; Christin
et al., 2008). It was long believed that a decline in
atmospheric CO2 concentrations triggered this showcase of
convergent evolution, but this has recently been questioned
because geological evidence for such a decline is missing
(Osborne and Beerling, 2006). As expected for a biochemical
pathway that independently evolved so many times, isoforms for the genes encoding C4 enzymes are already
present in C3 plants, but usually expressed at low levels and
with little regulation (Ku et al., 1996). C4 isogenes instead
are typically expressed at very high levels and regulated by
multiple stimuli (Sheen, 1999; Hibberd and Covshoff, 2010).
First, C4 gene expression has to be confined to the
mesophyll or bundle sheath, respectively, to avoid futile
cycling in one single tissue. Second, expression of C4 genes
and the activity of the encoded proteins are regulated by
light as observed for most other photosynthetic genes. Due
to their high abundance, the amount of C4 enzymes is, in
addition, often controlled by metabolic stimuli and the
availability of nutrients to ensure high resource-use efficiency. Sensing of intracellular sugar concentrations and
regulation by the availability of inorganic nitrogen are
important examples for this kind of regulation (Sheen,
1990; Suzuki et al., 1994; Offermann et al., 2008).
Certainly, the best understood part of the C4 syndrome is
C4 carbon cycling. It is therefore sensible to start engineering efforts with the transfer of genes encoding the enzymes
of the C4 photosynthetic cycle to C3 plants. The open
question is whether these genes will encode functional active
proteins showing the correct expression patterns and
responses to environmental stimuli in the foreign host.
During the last 20 years, expression of C4 enzymes in C3
plants has been repeatedly tested by several groups and,
although none of the approaches was successful in enhancing the photosynthetic properties of transgenic plants,
expression patterns and activities of the introduced genes
and the encoded proteins were studied in detail and provide
important lessons for future attempts. However, independent from the selection of promoters and genes, establishment of C4 metabolism in C3 crops is also an unprecedented
technological challenge in view of the number of genes that
will have to be transferred. Both gene selection and transfer
strategies are discussed in this review.
Choice of regulatory sequences
Early studies on the overexpression of C4 genes in C3
plants mostly made use of the constitutively expressed 35S
promoter from cauliflower mosaic virus. However, these
studies aimed at analysing the function of C4 proteins in
a C3 cellular environment (see also below) or to establish
a C4-like cycle in a single cell without differentiation in two
different cell types (Haeusler et al., 2002; Taniguchi et al.,
2008). This differentiation is probably required for the
optimal function of a C4 cycle in a C3 crop, although few
dicot species developed specialized C4 mechanisms where C4
enzymes are not distributed over two cell types but to
different specialized regions of a single cell (Edwards et al.,
2004). Constitutive promoter control is therefore probably
not a viable choice for C4 genes. In C4 plants, accumulation
of RNAs for C4 genes is mostly regulated on two different
levels: The activity of the corresponding promoter and posttranscriptional mechanisms that control the amount of
translatable RNA. Regulatory sequences therefore include
both elements upstream and downstream of the transcription initiation site. An excellent overview about the
regulation of C4 gene expression has recently been provided
by Hibberd and Covshoff (2010). Therefore, only data that
are relevant for the transfer of C4 genes to C3 plants are
discussed here in more detail.
Regulation of the C4-specific Pepc1 gene in maize is
a typical example of predominant promoter control. RNA
synthesis is restricted to leaves and within the leaves to
mesophyll cells. The establishment of this expression
pattern has been associated with particular, developmentally regulated histone modifications on the promoter
(Danker et al., 2008; Offermann et al., 2008), but the precise
cis-acting elements are unknown. For the analogous PpcA
promoter in Flaveria trinervia, a specific promoter domain
has been mapped that is necessary for the restriction of
promoter activity to the mesophyll (Gowik et al., 2004;
Akyildiz et al., 2007). Activity of the maize Pepc1 promoter
is enhanced by light, and transcription factors of the DOF
family that bind to specific promoter elements have been
implicated in the control of this process (Yanagisawa and
Sheen, 1998; Cavalar et al., 2003). Thus, the two most
critical properties of Pepc gene expression are controlled on
this level. Fortunately, both properties were recapitulated
when the intact Pepc1 gene (containing regulatory and
coding sequences) was transferred to rice. The C4 gene was
expressed in the C3 mesophyll at high levels and in a lightresponsive manner (Ku et al., 1999; Kausch et al., 2001).
There is evidence that some of the transcription factors
binding to the Pepc1 promoter of maize are also conserved
in rice (Matsuoka et al., 1994). Similarly, the Flaveria
trinervia PpcA promoter functions as a strong palisade
parenchyma-specific promoter in the evolutionary distant
dicot species tobacco (Gowik et al., 2004). However, the
intact maize Pepc1 gene is not faithfully transcribed in
tobacco as transcription is initiated from false upstream
promoter positions (Hudspeth et al., 1992). Thus, the use of
endogenous C4 promoters in C3 plants seems to be
a straightforward strategy to control C4-like expression
patterns in C3 species if the evolutionary distance between
the donor and target species is limited. Promising results
similar to those obtained with Pepc1 were also described for
Transfer of C4 genes to C3 plants | 3013
the intact maize Ppdk gene in rice (Fukayama et al., 2001).
The gene was highly expressed and RNA accumulated in
response to light. A second construct that did not contain
introns and the endogenous terminator sequence showed
much lower expression, suggesting that RNA stability
contributes to the regulation of maize Ppdk, at least in rice
(Fukayama et al., 2001). The promoter including the 5#
untranslated region of the maize Ppdk gene was sufficient
for preferential expression of a reporter in mesophyll cells
and was inactive in roots or stems. As observed for Pepc1,
similar promoter segments were bound by transcription
factors in both maize and rice (Matsuoka et al., 1993).
Together, endogenous C4 promoters including untranslated regions are useful tools for the expression of C4 genes
in the mesophyll of related C3 plants (Fig. 1). However,
little is known about regulatory stimuli other than cell-type
specificity and expression in response to light. For instance,
expression of the Pepc1 gene in maize is strongly controlled
by the concentrations of various metabolites including
sugars (Sheen, 1990; Jang and Sheen, 1994; Kausch et al.,
2001). The availability of nitrate is transduced from root to
leaf by the cytokine Zeatin and directly affects RNA
synthesis from the Pepc1 promoter (Offermann et al., 2006;
Suzuki et al., 1994). Moreover, Pepc1 is under tight
circadian control in maize (Horst et al., 2009). All these
additional regulatory processes might be very important for
the economy of a C4 cycle in a C3 environment and,
therefore, for the achievable increases in yield potential.
These topics require more attention in future studies.
By contrast with mesophyll-specific genes that are often
controlled primarily on the level of promoter activity, posttranscriptional processes play a more important role in the
control of bundle sheath-specific gene expression in C4
plants. Confinement to the bundle sheath of the expression
of the genes encoding the small subunit of Rubisco (rbcS)
has been studied in most detail. In maize, promoter activity
as deduced from run-on analyses with isolated nuclei is
higher in bundle sheath cells compared with mesophyll cells
(Schaeffner and Sheen, 1991). This pattern might be
established dependent on illumination by binding of the
transcription factor TRM1 to both regions in the promoter
and the 3# untranslated regions (UTR) of the gene (Purcell
Fig. 1. Most promising strategies for the overexpression of C4
genes in mesophyll and bundle sheath cells of C3 plants. Efficient
expression of mesophyll-specific genes from C4 promoters has
been demonstrated, but the coding sequences might have to be
adapted to the C3 environment. Coding sequences from bundle
sheath-specific C4 genes tested so far resulted in highly active
proteins in the C3 environment, but gene regulation is complex and
might not be properly reproduced in C3 plants. Endogenous
bundle sheath-specific promoters provide a promising alternative.
et al., 1995; Xu et al., 2001). Similarly, bundle sheathspecific expression and light induction of the maize Me1
gene encoding C4-specific NADP-ME seems to be controlled by both transcriptional and post-transcriptional
processes (Sheen, 1999; Danker et al., 2008). In dicots such
as Flaveria and Amaranthus, both 5# UTR and 3# UTR
sequences are crucial for the control of bundle-sheath
specificity and light induction of rbcS genes by controlling
translation of unspecifically accumulating RNAs (Patel
et al., 2004, 2006). Similarly, NADP-Me genes in Flaveria
bidentis are seemingly controlled by both promoter sequences and UTRs (Marshall et al., 1997).
These complex regulatory patterns could hamper correct
expression of bundle sheath-specific C4 genes in C3 plants.
When the promoter and part of the coding region of the
maize Me1 gene were expressed in translational fusion to
b-glucuronidase in rice, the reporter accumulated in both
mesophyll and bundle sheath cells (Nomura et al., 2005a).
Expression of a similar construct with promoter and 5#
coding sequences from the Zoysia japonica PCK gene was
confined to the bundle sheath, but transcripts accumulated
to relatively low levels and independent of light (Nomura
et al., 2005a). The intact gene for aspartate aminotransferase from Panicum miliaceum showed neither bundle sheathspecificity nor light induction when placed in rice (Nomura
et al., 2005b). On the other hand, high level and bundlesheath specific expression of the Flaveria trinervia gene for
the P subunit of glycine decarboxylase can be recapitulated
in Arabidopsis (Engelmann et al., 2008). Further levels of
gene regulation, such as the strong metabolic control of
maize rbcS and Me1 (Sheen, 1990), have not been studied in
transgenic C3 plants so far.
Together, these data imply that it would be tricky to use
regulatory sequences from bundle sheath-specific C4 genes
for the establishment of a C4 cycle in C3 plants. The problem
could be solved by identification and co-transformation of
regulatory trans-acting factors from C4 plants. However, this
would considerably increase the number of genes that are
required to establish a C4 cycle in the C3 host (see also
below). Alternatively, a more extensive characterization of
additional bundle sheath-specific genes from C4 plants might
identify promoters or other regulatory sequences that also
control the desired expression patterns in foreign hosts.
Probably the most promising approach in my view is the
discovery of endogenous C3 plant promoters that control
high levels of bundle sheath-specific gene expression (Fig. 1).
C3 homologues of genes that have been co-opted into the C4
pathway during the evolution of C4 plants are primary
candidates for the identification of such regulatory sequences
(Brown et al., 2010).
Choice of coding sequences
Besides differences in the regulation of RNA synthesis and
stability, C4 genes also acquired changes in coding sequences that altered the activities of the encoded proteins. Again,
this has been best studied for PEPC. The C4 isoform
3014 | Peterhansel
possesses a high Km for the substrate PEP and a low
sensitivity to feedback inhibition by malate, while the C3
isoform shows opposite characteristics (Svensson et al.,
1997). Phosphorylation of the N-terminal domain of the C4
enzyme is important in the control of feedback inhibition
(Echevarrı́a et al., 1990). In maize, the N-terminus of C4
PEPC is phosphorylated in the light, but, in rice plants
overexpressing the C4 protein, phosphorylation occurs in
the dark (Fukayama et al., 2003). Suppression of PEPC
phosphorylation by pharmacological inhibition of de novo
protein synthesis resulted in reduced photosynthetic rates in
maize and sorghum (Bakrim et al., 1993). On the other
hand, antisense inhibition of PEPC kinase in Flaveria
bidentis neither affected photosynthetic performance nor
plant growth (Furumoto et al., 2007). Thus, the physiological role of this modification in vivo is still under debate. In
addition to N-terminal phosphorylation, C4 PEPC enzymes
also contain specific central domains and a typical serine in
the C-terminal part that are both important for allosteric
regulation (Blasing et al., 2000). Furthermore, there is
evidence for regulation of C4 PEPC by tethering of the
protein to membranes (Monreal et al., 2009).
Different PEPC proteins have been overproduced in C3
plants with the aim of establishing a C4-like cycle. In two
studies, bacterial enzymes from Synechococcus (Chen et al.,
2004) or Corynebacterium (Gehlen et al., 1996) were used
because these enzymes were expected to be active independent of post-translational modification and/or allosteric
regulation. In both cases, basal plant metabolism was
disturbed by the foreign protein suggesting that the enzymes
were active in vivo. Potato PEPC was engineered for high
substrate affinity and low product inhibition, properties
that are probably important for high protein activity in a C3
metabolic environment, and re-introduced into potato
plants. Characterization of the resulting transgenic lines
revealed redirection of carbon flow from sugars to amino
acids, reduced levels of phosphorylated intermediates, and
stunted growth (Rademacher et al., 2002). Similar modifications were also introduced into maize PEPC (Endo et al.,
2008), but experiments with transgenic plants have not been
published until today. By contrast, only minor effects on
plant metabolism were observed in rice plants overexpressing unmodified maize PEPC (Ku et al., 1999; Fukayama
et al., 2003). The data indicate that bacterial or engineered
PEPC enzymes are good starting points for the installation
of C4 metabolism in C3 plants (Fig. 1). It can be expected
that the deleterious effects of PEPC overexpression will be
diminished once the produced organic acids will be efficiently decarboxylated in a complete C4 cycle.
Towards the establishment of a more complete C4 cycle,
different decarboxylases were overproduced in rice chloroplasts. When maize NADP-ME was produced under the
control of a light-inducible (but not bundle sheath-specific)
rice promoter, high enzyme activities and a resulting
disturbance of the chloroplast redox balance were observed
(Takeuchi et al., 2000). Similarly, PCK from Urochloa
panicoides accumulated to high amounts of active enzyme
when targeted to rice chloroplasts (Suzuki et al., 2000).
Transgenic plants showed enhanced incorporation of CO2
into organic acids suggesting that overproduction of the
decarboxylase in the chloroplast even induced higher
activity of endogenous PEPC in the cytosol. The available
data imply that decarboxylases from C4 plants are highly
active in a C3 environment (Fig. 1).
In conclusion, there is no simple rule for the choice of
coding sequences. Dependent on the C3 background and the
C4 enzymes introduced, it will be necessary to test enzymes
with different regulatory properties. The most essential
properties and the corresponding post-translational modifications can be identified by studying the regulation of C4
enzymes in C4 plants. However, a C4 cycle in a C3 plant will
probably operate under different metabolic conditions. C4
plants evolved multiple secondary metabolic adaptations
that, most likely, will never be engineered in a C3 plant such
as the cell-type specific regulation of Calvin cycle activity,
nitrate and sulphate assimilation, photorespiration, and
other primary metabolic pathways (reviewed in Edwards
et al., 2001). Importantly, isolated candidate C4 enzymes
can not be tested in C3 plants because establishment of
partial C4 cycles will almost always result in deleterious
phenotypes as described above. Single-cell systems in the
mesophyll of C3 plants might be useful alternatives for such
tests until a first functional two-cell system has been
established. Inducible C4-like metabolism in the aquatic
plant Hydrilla verticillata provides a blueprint for a pathway
that pumps CO2 from the cytosol to the chloroplast within
one cell, but not between different cells (Magnin et al.,
1997). Such a cycle has been built up in rice by simultaneous overexpression of PEPC in the cytosol and
malate dehydrogenase, ME, and PPDK in the chloroplast
(Taniguchi et al., 2008). Although this transgenic pathway
is apparently not fully functional, it remains to be shown
whether improved versions of a single-cell system do not
provide plants with an advantage, at least under certain
growth conditions (Von Caemmerer, 2003). Independent of
this, the existing transgenic lines can be also used as an
interesting test system for future tailor-made coding sequences and proteins.
Multiple gene transfer
Transferring C4 metabolism and anatomy to rice is certainly
the most ambitious metabolic engineering approach ever. It
has recently been shown that hundreds of proteins differentially accumulate in maize mesophyll or bundle sheath
chloroplasts, respectively (Majeran et al., 2008; Majeran and
van Wijk, 2009). A comparison of the transcriptomes of the
closely related species Cleome spinosa (C3) and Cleome
gynandra (C4) revealed more than 600 differentially accumulating transcripts (Bräutigam et al., 2011). However, it is
probably not necessary to engineer all these adaptations into
rice to establish a functional C4 cycle. Some of the probably
most essential engineering steps are shown in Fig. 2. At least
five enzymes make up a C4 metabolic cycle in a NADP-malic
enzyme species, such as maize, that is mostly used as
Transfer of C4 genes to C3 plants | 3015
Fig. 2. Essential adaptations for the establishment of functional C4 metabolism in rice. Shown is a minimal C4 cycle together with
associated enzyme and transporter activities (bold): CA, b-carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; DiT1,
dicarboxylate transporter 1; MDH, malate dehydrogenase; DiT2, dicarboxylate transporter 2; ME, malic enzyme; MEP1, mesophyll envelope
protein 1; PPDK, pyruvate phosphate dikinase; PPT, phosphoenolpyruvate phosphate translocator. Three major adaptations of
endogenous processes are required (italics): The activity of the Calvin cycle in mesophyll chloroplasts has to be reduced, the number of
bundle sheath chloroplasts has to be enhanced, and the vein density has to be increased.
a blueprint for C4 rice. All these enzymes have posttranslational modifications with partially unknown importance (not shown). In addition, new transporters that are, in
part, poorly characterized are necessary to allow for the very
high metabolite fluxes into and out of the mesophyll and
bundle sheath chloroplasts (Bräutigam et al., 2008; Majeran
et al., 2008). Accurate function of C4 will also require celltype specific suppression of a number of endogenous genes.
The most important example is obviously restriction of
Rubisco expression to the high CO2 atmosphere in bundle
sheath cells. To this end, nuclear rbcS expression, as well as
expression of the large subunit of Rubisco from the
chloroplast genome, will have to be down-regulated in
mesophyll cells. Fortunately, there is evidence that reduced
abundance of the small subunit inhibits translation of the
RNA encoding the large subunit (Rodermel, 2001). Secondary cell-type specific biochemical adaptations of C4 plants
regarding assimilatory pathways or photorespiration, as
mentioned above, might not be absolutely necessary for C4
function. On the anatomical level, a reduction in bundle
density and an increase in bundle sheath chloroplast number
will be essential. While the genes controlling bundle density
in C4 plants are unknown, it has recently been shown that
overexpression of the transcription factor OsGLK1 is
sufficient to enhance the number of green chloroplasts in rice
vascular parenchyma and bundle sheath cells (Nakamura
et al., 2009). Other adaptations such as changes in chloroplast ultrastructure or positioning vary quite a lot between
different C4 subtypes (Dengler and Nelson, 1999; Mulhaidat
et al., 2007) and might not be absolutely required for
a functional C4 cycle.
Based on this rough and probably underestimating listing,
we might easily end up with 20 or more genes. To put this
number into context: the development of ‘Golden Rice’
varieties containing two transgenes that were sufficiently
optimized to be ready for field trials and trait introgression
into elite lines required more than 10 years (Al-Babili and
Beyer, 2005). More elaborate engineering approaches are still
in an experimental state and are being tested in model
species. On the records list of the most complex metabolic
engineering approaches in plants, two approaches were
found in Arabidopsis: (i) the transfer of six genes (including
resistance markers) for the production of a polyhydroxyalkanoate of commercial value (Slater et al., 1999) and (ii) the
establishment of a bypass reaction to photorespiration by the
step-wise transfer of eight genes (again including resistance
markers) (Kebeish et al., 2007). It is therefore obvious that
establishment of C4 rice will require more sophisticated gene
transfer technologies.
Multiple transgenes can be combined in a single plant by
genetic crossing of individual transgenic lines similar to
‘gene pyramiding’ strategies that are used in conventional
breeding (Ashikari and Matsuoka, 2006). Through repeated
crossings, an essentially unlimited number of transgene loci
can be combined in a single plant. However, the multiple
integration sites strongly impede the generation of homozygous lines and multiple transgene integration sites are not
compatible with current legal requirements for the release of
transgenic plants (Taverniers et al., 2008). Similar problems
occur if plants are sequentially transformed with different
vector constructs. In addition, the number of markers that
can be used for the selection of successful transformation
events is quite limited (Sundar and Sakthivel, 2008). In
contrast to these technologies, co-transformation of numerous vectors in a single transformation event surprisingly
often results in transgenic plants that integrated all the
transgenes at a single genomic locus (Agrawal et al., 2005;
Zhu et al., 2008a). There seems to be no bias for specific
sequences that are preferably integrated and many combinations of the different genes can be recovered from the
3016 | Peterhansel
transformation event. The number of genes that can be
transferred in a single transformation event is significant. In
rice, up to 11 different genes were integrated at one or two
loci and a subset of these genes was shown to be stably
expressed through generations (Afolabi et al., 2004). The
highest number reported to my knowledge is the successful
simultaneous transformation of 12 plasmids into the
genome of soybean suspension culture cells (Hadi et al.,
1996). A drawback of this technology is the frequent
occurrence of complex integration patterns at the target
locus including the integration of multiple copies of individual genes in tandem together with gene fragments of
unpredictable size (Pawlowski and Somers, 1996; Birch,
1997). It might be necessary to sequence a very high number
of integration events in order to isolate a locus exclusively
containing integrations of single copy full-length genes,
although some more recent attempts using linear DNA
fragments instead of circular plasmids resulted in higher
frequencies of simple genome integrations (Fu et al., 2000;
Lowe et al., 2009). Besides locus composition, it has to be
taken into consideration that multiple vector transfer is not
possible by Agrobacterium-mediated transformation, but
particle bombardment has to be used. However, the latter
is already the method of choice for the transformation of
most monocot crops (Barcelo and Lazzeri, 1995; Casas
et al., 1995). Therefore, co-transformation can be an
efficient method for the transfer of numerous genes when
large populations can be screened for the integration event
of choice. For a complex, but important trait such as C4
rice, extended screens for the perfect event would probably
be worthwhile.
An alternative to the transfer of multiple genes on
individual vectors is the assembly of single large-vector
molecules (Hamilton et al., 1996). Such a strategy is ideally
suited to generate ‘clean’ transgenics with a defined number
of transgenes that are integrated at a single locus. Vectors
such as binary bacterial artificial chromosomes (BiBAC)
and transformation-competent artificial chromosomes
(TAC) with the potential to accommodate hundreds of
kilobases of DNA were successfully transformed to rice by
either Agrobacterium-mediated transformation or particle
bombardment (Liu et al., 2002; He et al., 2003). However,
with few exceptions (e.g. Kubo et al., 2005), such large
DNA fragments were only used in methodological case
studies, but not for genetic engineering approaches. In even
more ambitious approaches, autonomous minichromosomes were designed that contain centromere and telomere
sequences as well as origins of replication (Carlson et al.,
2007; Yu et al., 2007; Birchler et al., 2008). The capacity of
such ‘plant artificial chromosomes’ is virtually unlimited
and the vector could be propagated as an independent
additional chromosome. Artificial chromosomes that contain megabasepairs of DNA were successfully propagated in
human cell cultures (Kuroiwa et al., 2000), but, in plants,
significant problems with inefficient transfer to daughter
cells during mitosis and meiosis were described (Houben
et al., 2008). Currently, this still hampers the application of
this most promising technology in rice genetic engineering.
Besides the challenges associated with the transfer of
large DNA fragments in an intact condition to plant
genomes, until recently it was impossible to assemble such
long artificial sequences. Two recent technological breakthroughs now allow for the design and synthesis of any
desired DNA molecule almost independent of its length:
First, recombination cloning enables the integration of
DNA sequences into vectors without the need to eliminate
specific restriction enzyme recognition sequences from the
cloned sequence (Karimi et al., 2007). The recombination
sites can be designed in a way that each recombination
event creates a new recombination target site that can be
used for the integration of the next gene. These multiround
recombination cloning strategies (Dafny-Yelin and Tzfira,
2007) allow for filling vectors up to their maximal capacity
with DNA molecules of any desired sequence if small
recombination sites between the different molecules are
acceptable. Second, and even more exciting, bottom-up
synthesis of very long DNA sequences is now possible.
Using a combination of chemical DNA synthesis, PCR
technologies, restriction and recombination cloning, the
complete >1 Megabasepair genome of a Mycoplasma
bacterium was recently artificially assembled (Gibson et al.,
2010).
These technological advances provide great promise that
creation of C4 rice will not fail because of challenges
associated with gene transfer. However, it is mandatory to
start significant additional efforts towards the further
development of routine technologies.
Acknowledgement
Work on the regulation of C4 genes in the author’s
laboratory has been continuously supported over more than
10 years by the Deutsche Forschungsgemeinschaft.
References
Afolabi AS, Worland B, Snape JW, Vain P. 2004. A large-scale
study of rice plants transformed with different T-DNAs provides new
insights into locus composition and T-DNA linkage configurations.
Theoretical and Applied Genetics 109, 815–826.
Agrawal P, Kohli A, Twyman R, Christou P. 2005. Transformation
of plants with multiple cassettes generates simple transgene
integration patterns and high expression levels. Molecular Breeding
16, 247–260.
Akyildiz M, Gowik U, Engelmann S, Koczor M, Streubel M,
Westhoff P. 2007. Evolution and function of a cis-regulatory module
for mesophyll-specific gene expression in the C4 dicot Flaveria
trinervia. The Plant Cell 19, 3391–3402.
Al-Babili S, Beyer P. 2005. Golden Rice: five years on the road—five
years to go? Trends in Plant Science 10, 565–573.
Ashikari M, Matsuoka M. 2006. Identification, isolation and
pyramiding of quantitative trait loci for rice breeding. Trends in Plant
Science 11, 344–350.
Transfer of C4 genes to C3 plants | 3017
Bakrim N, Prioul JL, Deleens E, Rocher JP, Arrio-Dupont M,
Vidal J, Gadal P, Chollet R. 1993. Regulatory phosphorylation of C4
phosphoenolpyruvate carboxylase (a cardinal event influencing the
photosynthesis rate in sorghum and maize). Plant Physiology 101,
891–897.
Barcelo P, Lazzeri PA. 1995. Transformation of cereals by
microprojectile bombardment of immature inflorescence and scutellum
tissues. Methods in Molecular Biology 49, 113–123.
Birch RG. 1997. Plant transformation: problems and strategies for
practical application. Annual Review of Plant Physiology and Plant
Molecular Biology 48, 297–326.
Birchler JA, Yu W, Han F. 2008. Plant engineered minichromosomes
and artificial chromosome platforms. Cytogenetic and Genome
Research 120, 228–232.
Blasing OE, Westhoff P, Svensson P. 2000. Evolution of C4
phosphoenolpyruvate carboxylase in Flaveria, a conserved serine
residue in the carboxyl-terminal part of the enzyme is a major
determinant for C4-specific characteristics. Journal of Biological
Chemistry 275, 27917–27923.
Bräutigam A, Hoffmann-Benning S, Weber APM. 2008.
Comparative proteomics of chloroplasts envelopes from C3 and C4
plants reveals specific adaptations of the plastid envelope to C4
photosynthesis and candidate proteins required for maintaining C4
metabolite fluxes. Plant Physiology 148, 568–579.
Bräutigam A, Kajala K, Wullenweber J, et al. 2011. An mRNA
blueprint for C4 photosynthesis derived from comparative transcriptomics
of closely related C3 and C4 species. Plant Physiology 155, 142–156.
Brown NJ, Palmer BG, Stanley S, et al. 2010. C4 acid
decarboxylases required for C4 photosynthesis are active in the midvein of the C3 species Arabidopsis thaliana, and are important in sugar
and amino acid metabolism. The Plant Journal 61, 122–133.
Carlson SR, Rudgers GW, Zieler H, Mach JM, Luo S, Grunden E,
Krol C, Copenhaver GP, Preuss D. 2007. Meiotic transmission of
an in vitro assembled autonomous maize minichromosome. PLoS
Genetics 3, e179.
Casas AM, Kononowicz AK, Bressan RA, Hasegawa PM. 1995.
Cereal transformation through particle bombardment. Plant Breeding
Reviews 13, 235–264.
Cavalar M, Möller C, Offermann S, Krohn NM, Grasser KD,
Peterhänsel C. 2003. The interaction of DOF transcription factors
with nucleosomes depends on the positioning of the binding site and
is facilitated by maize HMGB5. Biochemistry 42, 2149–2157.
Chen LM, Li KZ, Miwa T, Izui K. 2004. Overexpression of
a cyanobacterial phosphoenolpyruvate carboxylase with diminished
sensitivity to feedback inhibition in Arabidopsis changes amino acid
metabolism. Planta 219, 440–449.
Christin P-A, Besnard G, Samaritani E, Duvall MR, Hodkinson TR,
Savolainen V, Salamin N. 2008. Oligocene CO2 decline promoted C4
photosynthesis in grasses. Current Biology 18, 37–43.
Danker T, Dreesen B, Offermann S, Horst I, Peterhänsel C.
2008. Developmental information but not promoter activity controls the
methylation state of histone H3 lysine 4 on two photosynthetic genes
in maize. The Plant Journal 53, 465–474.
Dengler NG, Nelson T. 1999. Leaf structure and development in C4
plants. In: Sage RF, Monson RK, eds. C4 plant biology. San Diego,
USA: Academic Press, 133–172.
Echevarrı́a C, Vidal J, Jiao J, Chollet R. 1990. Reversible light
activation of the phosphoenolpyruvate carboxylase protein-serine
kinase in maize leaves. FEBS Letters 275, 25–28.
Edwards GE, Franceschi VR, Ku MSB, Voznesenskaya EV,
Pyankov VI, Andreo CS. 2001. Compartmentation of photosynthesis
in cells and tissues of C4 plants. Journal of Experimental Botany 52,
577–590.
Edwards GE, Franceschi VR, Voznesenskaya EV. 2004. Singlecell C4 photosynthesis versus the dual-cell (Kranz) paradigm. Annual
Review of Plant Biology 55, 173–196.
Endo T, Mihara Y, Furumoto T, Matsumura H, Kai Y, Izui K.
2008. Maize C4-form phosphoenolpyruvate carboxylase engineered to
be functional in C3 plants: mutations for diminished sensitivity to
feedback inhibitors and for increased substrate affinity. Journal of
Experimental Botany 59, 1811–1818.
Engelmann S, Wiludda C, Burscheidt J, Gowik U, Schlue U,
Koczor M, Streubel M, Cossu R, Bauwe H, Westhoff P. 2008.
The gene for the P-subunit of glycine decarboxylase from the C4
species Flaveria trinervia: analysis of transcriptional control in
transgenic Flaveria bidentis (C4) and Arabidopsis (C3). Plant Physiology
146, 1773–1785.
Fu X, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D,
Twyman RM, Christou P, Kohli A. 2000. Linear transgene
constructs lacking vector backbone sequences generate low-copynumber transgenic plants with simple integration patterns. Transgenic
Research 9, 11–19.
Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S,
Furbank RT, Miyao M. 2003. Activity regulation and physiological
impacts of maize C4-specific phosphoenolpyruvate carboxylase
overproduced in transgenic rice plants. Photosynthesis Research 77,
227–239.
Fukayama H, Tsuchida H, Agarie S, et al. 2001. Significant
accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3
plant, rice. Plant Physiology 127, 1136–1146.
Furumoto T, Izui K, Quinn V, Furbank RT, von Caemmerer S.
2007. Phosphorylation of phosphoenolpyruvate carboxylase is not
essential for high photosynthetic rates in the C4 species Flaveria
bidentis. Plant Physiology 144, 1936–1945.
Gehlen J, Panstruga R, Smets H, et al. 1996. Effects of altered
phosphoenolpyruvate carboxylase activities on transgenic C3 plant
Solanum tuberosum. Plant Molecular Biology 32, 831–848.
Dafny-Yelin M, Tzfira T. 2007. Delivery of multiple transgenes to
plant cells. Plant Physiology 145, 1118–1128.
Gibson DG, Glass JI, Lartigue C, et al. 2010. Creation of a bacterial
cell controlled by a chemically synthesized genome. Science 329,
52–56.
Dai Z, Ku M, Edwards GE. 1993. C4 photosynthesis (the CO2concentrating mechanism and photorespiration. Plant Physiology 103,
83–90.
Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M,
Streubel M, Westhoff P. 2004. Cis-regulatory elements for
mesophyll-specific gene expression in the C4 plant Flaveria trinervia,
3018 | Peterhansel
the promoter of the C4 phosphoenolpyruvate carboxylase gene. The
Plant Cell 16, 1077–1090.
Hadi MZ, McMullen MD, Finer JJ. 1996. Transformation of 12
different plasmids into soybean via particle bombardment. Plant Cell
Reports 15, 500–505.
Haeusler RE, Hirsch HJ, Kreuzaler F, Peterhänsel C. 2002.
Overexpression of C4-cycle enzymes in transgenic C3 plants:
a biotechnological approach to improve C3-photosynthesis. Journal of
Experimental Botany 53, 591–607.
Hamilton CM, Frary A, Lewis C, Tanksley SD. 1996. Stable
transfer of intact high molecular weight DNA into plant chromosomes.
Proceedings of the National Academy of Sciences. USA 93,
9975–9979.
He R-F, Wang Y, Shi Z, Ren X, Zhu L, Weng Q, He G- C. 2003.
Construction of a genomic library of wild rice and Agrobacteriummediated transformation of large insert DNA linked to BPH resistance
locus. Gene 321, 113–121.
Hibberd JM, Covshoff S. 2010. The regulation of gene expression
required for C4 photosynthesis. Annual Review of Plant Biology 61,
181–207.
Hibberd JM, Sheehy JE, Langdale JA. 2008. Using C4
photosynthesis to increase the yield of rice-rationale and feasibility.
Current Opinion in Plant Biology 11, 228–231.
Horst I, Offermann S, Dreesen B, Niessen M, Peterhänsel C.
2009. Core promoter acetylation is not required for high transcription
from the phosphoenolpyruvate carboxylase promoter in maize.
Epigenetics Chromatin 2, 17.
Houben A, Dawe RK, Jiang J, Schubert I. 2008. Engineered plant
minichromosomes: a bottom-up success? The Plant Cell 20, 8–10.
Hudspeth RL, Grula JW, Dai Z, Edwards GE, Ku MSB. 1992.
Expression of maize phospoenolpyruvate carboxylase in transgenic
tobacco. Plant Physiology 98, 458–464.
Jang JC, Sheen J. 1994. Sugar sensing in higher plants. The Plant
Cell 6, 1665–1679.
Kanai R, Edwards GE. 1999. The biochemistry of C4 photosynthesis.
In: Sage RF, Monson RK, eds. C4 plant biology. San Diego: Academic
Press, 49–87.
Karimi M, Depicker A, Hilson P. 2007. Recombinational cloning
with plant Gateway vectors. Plant Physiology 145, 1144–1154.
Kausch AP, Owen TP, Zachwieja SJ, Flynn AR, Sheen J. 2001.
Mesophyll-specific, light and metabolic regulation of the C4 PPCZm1
promoter in transgenic maize. Plant Molecular Biology 45, 1–15.
Kebeish R, Niessen M, Thiruveedhi K, Bari R, Hirsch H-J,
Rosenkranz R, Stäbler N, Schönfeld B, Kreuzaler F,
Peterhänsel C. 2007. Chloroplastic photorespiratory bypass
increases photosynthesis and biomass production in Arabidopsis
thaliana. Nature Biotechnology 25, 593–599.
Ku M, Kano-Murakami Y, Matsuoka M. 1996. Evolution and
expression of C4 photosynthesis genes. Plant Physiology 111,
949–957.
Ku MS, 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 transgenic
rice plants. Nature Biotechnology 17, 76–80.
Kubo A, Rahman S, Utsumi Y, et al. 2005. Complementation of
sugary-1 phenotype in rice endosperm with the wheat isoamylase1
gene supports a direct role for isoamylase1 in amylopectin
biosynthesis. Plant Physiology 137, 43–56.
Kuroiwa Y, Tomizuka K, Shinohara T, Kazuki Y, Yoshida H,
Ohguma A, Yamamoto T, Tanaka S, Oshimura M, Ishida I. 2000.
Manipulation of human minichromosomes to carry greater than
megabase-sized chromosome inserts. Nature Biotechnology 18,
1086–1090.
Liu Y-G, Liu H, Chen L, et al. 2002. Development of new
transformation-competent artificial chromosome vectors and rice
genomic libraries for efficient gene cloning. Gene 282, 247–255.
Lowe B, Shiva Prakash N, Way M, Mann M, Spencer T,
Boddupalli R. 2009. Enhanced single copy integration events in corn
via particle bombardment using low quantities of DNA. Transgenic
Research 18, 831–840.
Magnin NC, Cooley BA, Reiskind JB, Bowes G. 1997. Regulation
and localization of key enzymes during the induction of Kranz-less, C4type photosynthesis in Hydrilla verticillata. Plant Physiology 115,
1681–1689.
Majeran W, van Wijk KJ. 2009. Cell-type-specific differentiation of
chloroplasts in C4 plants. Trends in Plant Science 14, 100–109.
Majeran W, Zybailov B, Ytterberg AJ, Dunsmore J, Sun Q, van
Wijk KJ. 2008. Consequences of C4 differentiation for chloroplast
membrane proteomes in maize mesophyll and bundle sheath cells.
Molecular Cell Proteomics 7, 1609–1638.
Marshall JS, Stubbs JD, Chitty JA, Surin B, Taylor WC. 1997.
Expression of the C4 Me1 gene from Flaveria bidentis requires an
interaction between 5# and 3# sequences. The Plant Cell 9,
1515–1525.
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). The Plant
Journal 6, 311–319.
Matsuoka M, Tada Y, Fujimura T, Kano-Murakami Y. 1993.
Tissue-specific light-regulated expression directed by the promoter of
a C4 gene, maize pyruvate, orthophosphate dikinase, in a C3 plant,
rice. Proceedings of the National Academy of Sciences. USA 90,
9586–9590.
Maurino VG, Peterhänsel C. 2010. Photorespiration: current status
and approaches for metabolic engineering. Current Opinion in Plant
Biology 13, 249–256.
Mitchell PL, Sheehy JE. 2006. Supercharging rice photosynthesis to
increase yield. New Phytologist 171, 688–693.
Monreal JA, McLoughlin F, Echevarria C, Garcia-Maurino S,
Testerink C. 2009. Phosphoenolpyruvate carboxylase from C4 leaves
is selectively targeted for inhibition by anionic phospholipids. Plant
Physiology 152, 634–638.
Mulhaidat R, Sage R, Dengler N. 2007. Diversity of kranz anatomy and
biochemistry in C4 eudicots. American Journal of Botany 94, 362–381.
Nakamura H, Muramatsu M, Hakata M, Ueno O, Nagamura Y,
Hirochika H, Takano M, Ichikawa H. 2009. Ectopic overexpression
of the transcription factor OsGLK1 induces chloroplast development in
non-green rice cells. Plant and Cell Physiology 50, 1933–1949.
Transfer of C4 genes to C3 plants | 3019
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 and Cell Physiology 46, 754–761.
Nomura M, Higuchi T, Katayama K, Taniguchi M, MiyaoTokutomi M, Matsuoka M, Tajima S. 2005b. The promoter for C4type mitochondrial aspartate aminotransferase does not direct bundle
sheath-specific expression in transgenic rice plants. Plant and Cell
Physiology 46, 743–753.
Offermann S, Danker T, Dreymüller D, Kalamajka R, Töpsch S,
Weyand K, Peterhänsel C. 2006. Illumination is necessary and
sufficient to induce histone acetylation independent of transcriptional
activity at the C4-specific phosphoenolpyruvate carboxylase promoter
in maize. Plant Physiology 141, 1078–1088.
Offermann S, Dreesen B, Horst I, Danker T, Jaskiewicz M,
Peterhänsel C. 2008. Developmental and environmental signals
induce distinct histone acetylation profiles on distal and proximal
promoter elements of the C4-Pepc gene in maize. Genetics 179,
1891–1901.
Sheen J. 1999. C4 gene expression. Annual Review of Plant
Physiology and Plant Molecular Biology 50, 187–217.
Slater S, Mitsky TA, Houmiel KL, et al. 1999. Metabolic
engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrateco-3-hydroxyvalerate) copolymer production. Nature Biotechnology
17, 1011–1016.
Sundar IK, Sakthivel N. 2008. Advances in selectable marker genes
for plant transformation. Journal of Plant Physiology 165, 1698–1716.
Suzuki I, Cretin C, Omata T, Sugiyama T. 1994. Transcriptional
and posttranscriptional regulation of nitrogen-responding expression
of phosphoenolpyruvate carboxylase gene in maize. Plant Physiology
105, 1223–229.
Suzuki S, Murai N, Burnell JN, Arai M. 2000. Changes in
photosynthetic carbon flow in transgenic rice plants that express C4type phosphoenolpyruvate carboxykinase from Urochloa panicoides.
Plant Physiology 124, 163–172.
Svensson P, Biasing OE, Westhoff P. 1997. Evolution of the
enzymatic characteristics of C4 phosphoenolpyruvate carboxylase.
European Journal of Biochemistry 246, 452–460.
Osborne CP, Beerling DJ. 2006. Nature’s green revolution: the
remarkable evolutionary rise of C4 plants. Philosophical Transactions
of the Royal Society B: Biological Sciences 361, 173–194.
Takeuchi Y, Akagi H, Kamasawa N, Osumi M, Honda H. 2000.
Aberrant chloroplasts in transgenic rice plants expressing a high level
of maize NADP-dependent malic enzyme. Planta 211, 265–274.
Patel M, Corey AC, Yin L-P, Ali S, Taylor WC, Berry JO. 2004.
Untranslated regions from C4 Amaranth AhRbcS1 mRNAs confer
translational enhancement and preferential bundle sheath cell
expression in transgenic C4 Flaveria bidentis. Plant Physiology 136,
3550–3561.
Taniguchi Y, Ohkawa H, Masumoto C, et al. 2008. Overproduction
of C4 photosynthetic enzymes in transgenic rice plants: an approach
to introduce the C4-like photosynthetic pathway into rice. Journal of
Experimental Botany 59, 1799–1809.
Patel M, Siegel AJ, Berry JO. 2006. Untranslated regions of
FbRbcS1 mRNA mediate bundle sheath cell-specific gene expression
in leaves of a C4 plant. Journal of Biological Chemistry 281,
25485–25491.
Pawlowski W, Somers D. 1996. Transgene inheritance in plants
genetically engineered by microprojectile bombardment. Molecular
Biotechnology 6, 17–30.
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. Proceedings of the National
Academy of Sciences. USA 92, 11504–11508.
Rademacher T, Häusler RE, Hirsch HJ, Zhang L, Lipka V,
Weier D, Kreuzaler F, Peterhänsel C. 2002. An engineered
phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow
in transgenic potato plants. The Plant Journal 32, 25–39.
Rodermel S. 2001. Pathways of plastid-to-nucleus signaling. Trends
in Plant Science 6, 471–478.
Sage RF. 2004. The evolution of C4 photosynthesis. New Phytologist
161, 341–370.
Schaeffner AR, Sheen J. 1991. Maize rbcS promotor activity
depends on sequence elements not found in dicot rbcS promotors.
The Plant Cell 3, 997–1012.
Sheen J. 1990. Metabolic repression of transcription in higher plants.
The Plant Cell 2, 1027–1038.
Taverniers I, Papazova N, Bertheau Y, De Loose M, HolstJensen A. 2008. Gene stacking in transgenic plants: towards
compliance between definitions, terminology, and detection within the EU
regulatory framework. Environmental Biosafety Research 7, 197–218.
Von Caemmerer S. 2003. C4 photosynthesis in a single C3 cell is
theoretically inefficient but may ameliorate internal CO2 diffusion
limitations of C3 leaves. Plant. Cell and Environment 26, 1191–1197.
Xu T, Purcell M, Zucchi P, Helentjaris T, Bogorad L. 2001. TRM1,
a YY1-like suppressor of rbcS-m3 expression in maize mesophyll cells.
Proceedings of the National Academy of Sciences. USA 98,
2295–2300.
Yanagisawa S, Sheen J. 1998. Involvement of maize Dof zinc finger
proteins in tissue-specific and light-regulated gene expression. The
Plant Cell 10, 75–89.
Yu W, Han F, Gao Z, Vega JM, Birchler JA. 2007. Construction
and behavior of engineered minichromosomes in maize. Proceedings
of the National Academy of Sciences. USA 104, 8924–8929.
Zhu C, Naqvi S, Breitenbach J, Sandmann G, Christou P,
Capell T. 2008a. Combinatorial genetic transformation generates
a library of metabolic phenotypes for the carotenoid pathway in maize.
Proceedings of the National Academy of Sciences. USA 105,
18232–18237.
Zhu X-G, Long SP, Ort DR. 2008b. What is the maximum efficiency
with which photosynthesis can convert solar energy into biomass?
Current Opinion in Biotechnology 19, 153–159.