Urte Schlüter and Andreas P.M. Weber*
Institute of Plant Biochemistry and Cluster of Excellence on Plant Science (CEPLAS), Heinrich-Heine-University, Universitätsstrasse 1, D-40225 Düsseldorf,
Germany
*Corresponding author: E-mail, [email protected]; Fax, +49 (0)211 8113706.
(Received November 2, 2015; Accepted January 5, 2016)
C4 photosynthesis enables high photosynthetic energy conversion efficiency as well as high nitrogen and water use efficiencies. Given the multitude of biochemical, structural and
molecular changes in comparison with C3 photosynthesis, it
appears unlikely that such a complex trait would evolve in a
single step. C4 photosynthesis is therefore believed to have
evolved from the ancestral C3 state via intermediary stages.
Consequently, the identification and detailed characterization
of plant species representing transitory states between C3 and
C4 is important for the reconstruction of the sequence of
evolutionary events, especially since C4 evolution occurred in
very different phylogenetic backgrounds. There is also significant interest in engineering of C4 or at least C4-like elements
into C3 crop plants. A detailed and mechanistic understanding
of C3–C4 intermediates is likely to provide guidance for the
experimental design of such approaches. Here we provide an
overview on the most relevant results obtained on C3–C4
intermediates to date. Recent knowledge gains in this field
will be described in more detail. We thereby concentrate especially on biochemical and physiological work. Finally, we will
provide a perspective and outlook on the continued importance of research on C3–C4 intermediates.
Keywords: C4 photosynthesis C3–C4 intermediate Evolution Photorespiration.
Abbreviations: CBC, Calvin–Benson cycle; BS, bundle sheath;
GDC, glycine decarboxylase complex; M, mesophyll; NUE,
nitrogen use efficiency; PGA, 3-phosphoglycerate; RuBisCO,
ribulose-1,5-bisphosphate
carboxylase/oxygenase;
WUE,
water use efficiency; PEPC, phosphoenolpyruvate carboxylase;
2PG, 2-phosphoglyceric acid.
A Historical Perspective on the Discovery of
C3–C4 Intermediate Species
The main principle of C4 photosynthesis was reported 50 years
ago by Hatch and Slack (1966): after short-term exposure of
illuminated sugarcane leaf segments to 14CO2 for 1 s, the vast
majority of the label (93%) was detected in C4 dicarboxylic acids
(malate, aspartate and oxaloacetate), and not in 3-phosphogylceric acid (3PGA), as would be the case in C3 plants. After 20 s,
approximately 30% of the 14C-label was detectable in 3PGA,
whereas the proportion of label in C4 acids declined, which
indicated that the radiolabeled carbon was transferred via a
C4 compound to the classical C3 Calvin–Benson cycle (CBC).
Later work revealed that C4 photosynthesis involves a spatial
separation of the two consecutive carboxylation reactions:
After primary fixation of carbon by phosphoenolpyruvate carboxylase (PEPC), the resulting C4 compound is transported to
the site of the ribulose 1,5-bisphosphate carboxylase/oxygenase
(RuBisCO) reaction, where CO2 is released again by decarboxylation of the C4 acid (Hatch 1987; Fig. 1D). By concentrating
CO2 at the site of RuBisCO via the C4 biochemical carbon
pump, the oxygenation reaction of RuBisCO, which leads to
the formation of the toxic metabolic intermediate 2-phosphoglyceric acid (2PG), is suppressed. Detoxifying 2PG requires the
photorespiratory cycle, which demands energy and leads to the
loss of previously fixed CO2. The high CO2 concentration
around RuBisCO achieved through C4 photosynthesis suppresses the photorespiratory cycle, a situation that enables
higher photosynthetic efficiency in C4 plants and hence frequently also higher productivity than in their ancestral C3 relatives, especially in sunny, hot and dry climates.
Within the angiosperms, C4 photosynthesis evolved independently >60 times (Sage et al. 2012). In the majority of C4
plants, the C4 carbon-concentrating mechanism and the CBC
are distributed over two distinct leaf cell types, the mesophyll
(M) and the bundle sheath (BS) cells, with a few exceptions that
employ a single-celled C4 mechanism (Voznesenskaya et al.
2001). Reaping the benefits of C4 photosynthesis therefore depends on numerous leaf anatomical and biochemical re-adjustments. Traditionally, C4 plants are distinguished from C3 plants
by the following characteristic features: (i) short-term labeling
with 14C leads to initial incorporation of the label into the C4
compounds oxaloacetate, malate and, frequently also, aspartate; (ii) a very low CO2 compensation point; (iii) reduced discrimination against the heavy carbon isotope 13C; (iv)
anatomical changes associated with narrow vein spacing and
increased numbers of chloroplasts in enlarged BS cells; (v) high
abundance and cell-specific expression of C4 shuttle enzymes;
and (vi) reduced abundance and altered cell-specific expression
of CBC and photorespiratory enzymes (reviewed in Sage et al.
2012).
Despite the frequent convergent evolution of C4 species
from C3 ancestors, it is unlikely that such a complex trait
would have evolved in just one single giant step. The existence
Plant Cell Physiol. 57(5): 881–889 (2016) doi:10.1093/pcp/pcw009, Advance Access publication on 17 February 2016,
available online at www.pcp.oxfordjournals.org
! The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Special Focus Issue – Mini Review
The Road to C4 Photosynthesis: Evolution of a Complex
Trait via Intermediary States
U. Schlüter and A. P. M. Weber | The road to C4
Fig. 1 Evolution from C3 to C4 photosynthesis. (A) C3 photosynthesis: the Calvin–Benson cycle and photorespiration work independently in
both cell types. (B) Basic or early intermediate photosynthesis: the activity of the GDC has shifted towards the bundle sheath, activating CO2
transport from the mesophyll to the bundle sheath by a photorespiratory glycine pump. In additional to CO2, the GDC reaction also releases
ammonium and consequently motivates N balancing mechanisms between the cells. This can be realized by transport of different metabolites;
the model of Mallmann et al. (2014) suggests an alanine–pyruvate shuttle as one of the most likely solutions (the oxygenase reaction of Rubisco
(continued)
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Plant Cell Physiol. 57(5): 881–889 (2016) doi:10.1093/pcp/pcw009
of evolutionary intermediate forms was therefore expected and,
indeed, in 1974, Kennedy and Laetsch presented Mollugo verticillata as the first candidate for a C3–C4 intermediate photosynthesis type (Kennedy and Laetsch 1974). The general leaf
anatomy of M. verticillata appeared to be typical C3, but the BS
cells had numerous, well-developed chloroplasts similar to the
typical picture in C4. Photorespiratory rates of M. verticillata
were in between the values of C3 and C4 species. Exposure to
14
CO2 resulted in almost equal labeling of C3 and C4 compounds as primary photosynthetic products.
At the time of initial discovery, the mechanisms enabling a
mixture of C3- and C4-related features in M. verticillata were far
from understood, and the assignment of this species as intermediary between C3 and C4 photosynthesis was merely a hypothesis. The interest in the discovery of possible C3–C4
intermediates was, however, stimulated, in particular because
it was very clear from the beginning that knowledge gained
from the study of intermediates would enable an understanding of the mechanisms underlying the evolution of C4 photosynthesis. In the years following the description of
M. verticillata, many more potential C3–C4 intermediates
have been identified and studied. A survey of CO2 compensation points in several hundred eudicot and monocot species
identified the Brassicaceae Moricandia arvensis and the grass
specie Panicum milioides (= Steinchisma hians) as further C3–C4
intermediates (Krenzer et al. 1975). A comprehensive review of
C3–C4 intermediates published in 1987 (Edwards and Ku 1987)
already listed 22 potential C3–C4 species, most of them belonging to orders which also included C4 species. Investigation of
this increased variety of intermediates permitted the definition
of common distinctive features: (i) a reduced CO2 compensation point; (ii) a reduced sensitivity of photosynthesis to O2;
and (iii) BS cells with an increased number of centripetally
arranged chloroplasts and mitochondria, as compared with
C3 species. However, the d13C values of C3–C4 intermediates
were found to be close to those of C3 species, which clearly
distinguishes the intermediates from bona fide C4 plants
(Edwards and Ku 1987). On the basis of the observed reduction
in the apparent rate of photorespiration and high rates of lightdependent 14C labeling of glycine, Monson, Edwards and Ku
(1984) proposed that glycine is transported from the M to the
BS cells, where CO2 released from the glycine decarboxylase
reaction would be refixed by the CBC, thereby establishing a
glycine-based mechanism for pumping carbon into BS cells
(Monson et al. 1984, Edwards and Ku, 1987). This model implies
that the majority of glycine-decarboxylating activity in leaves
would be located in BS cells, and not in M cells, as it is typical for
C3 species.
Experimental support for the proposed confinement of the
glycine decarboxylase reaction to the BS cells was later provided
in the intermediate M. arvensis by immunolocalization as well
as enzyme activity assays in M- and BS-enriched extracts
(Rawsthorne et al. 1988a, Rawsthorne et al. 1988b). These results led to the first experimentally confirmed C3–C4 photosynthesis model (Rawsthorne et al. 1988b). Absence of a
functioning glycine decarboxylase complex (GDC) in the M
cells causes glycine accumulation in the M, which drives diffusion of glycine to the BS, where its oxidative decarboxylation by
the mitochondrial GDC system produces CO2 (Fig. 1B). The
close association of mitochondria and chloroplasts at the inner
wall of BS cells enhances the potential for re-assimilation of
photorespiratory CO2, while at the same time increasing the
concentration of CO2 at the site of RuBisCO in BS cells. The
products of the decarboxylation reaction, in particular serine,
then need to move back to the M cells for re-establishment of
C and N balance between the cells (Rawsthorne et al. 1988b).
The model proposed by Rawsthorne and colleagues could explain the reduction of the CO2 compensation point observed in
C3–C4 intermediates, even in the absence of an upstream C4
cycle. The confinement of the GDC activity to the BS in C3–C4
intermediates was later confirmed in a variety of independently
evolved C3–C4 intermediate species from very different phylogenetic backgrounds, including Panicum milioides, M. verticillata, different Flaveria species (Hylton et al. 1988), Diplotaxis
tenuifolia (Ueno et al. 2006), Brassica gravinae (Ueno 2011),
Salsola divaricata (Gandin et al. 2014), Heliotropium convolvulaceum and H. greggii (Muhaidat et al. 2011), Euphorbia acuta
(T.L. Sage et al. 2011) and Portulaca cryptopetala
(Voznesenskaya et al. 2010). The BS-specific activity of the
GDC appears to be a common feature that C3–C4 intermediates share with bona fide C4 species, which supports the placement of intermediates as stations on the evolutionary road
from C3 to C4 (Rawsthorne et al. 1988b).
In addition to this feature, that is common to all C3-C4
intermediate species studied to date, gradual differences were
observed for the presence of other C4-like features in various
intermediates (Ku et al. 1983, Monson et al. 1986). This includes
especially the abundance and cell specificity of PEPC and other
typical C4 enzymes, the ratio between primary 14C label recovered in C3 and C4 compounds, and a reduction in vein
spacing. Analysis of these features in different C3–C4 intermediates led to the definition of the sequence of key events on the
road from C3 to C4: (i) confinement of the GDC to the BS; (ii)
increase of PEPC and C4 cycle activity; (iii) complete shift of
RuBisCO activity to the BS cells; and (iv) finally the optimization
of the system (Monson and Rawsthorne 2000, Sage 2004, Gowik
in the bundle sheath is continuously reduced and is omitted in the drawing for reasons of clarity in B–D). (C) Advanced intermediate
photosynthesis: any increase in PEPC activity of the mesophyll will produce C4 metabolites which can be easily integrated into the photorespiratory shuttle; further parallel increases in PEPC and C4 shuttle activity can further contribute to plant fitness (see Mallmann et al. 2014). (D)
C4 photosynthesis: RuBisCO activity shifted completely to the bundle sheath; the figure demonstrates the NADP-malic enzyme (NADP-ME)
subtype. Plant species operating the described photosynthesis systems are given at the side. Classification is according to Hatterley et al. (1986),
Edwards and Ku (1987), Brown and Morgan (1980) and Muhaidat et al. (2011). Abbreviations: GDC, glycine decarboxylase complex; RUBISCO,
ribulose-1,5-bisphosphate carboxylase/oxygenase; PEP, phosphoenolpyruvate, PEPC. PEP carboxylase; 3PGA. 3-phosphoglycerate; 2PG, 2-phosphoglycolate; OAA, oxaloacetic acid; CB cycle, Calvin–Benson cycle.
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U. Schlüter and A. P. M. Weber | The road to C4
and Westhoff 2011, Leegood 2013). In some studies, these
changes are also associated with the definition of different
C3–C4 subtypes: type I intermediates possess a glycine pump
but show no changes in apparent PEPC activity (Fig. 1B); while
in type II plants enhanced PEPC activity induces a limited C4
cycle (Fig. 1C; Edwards and Ku 1987). Additionally, a C4-like
phenotype was defined in Flaveria brownii, a species with a
strong C4 cycle, but still with remaining RuBisCO activity in
the mesophyll (Brown et al. 1993).
The entire spectrum of sequential inclusion of C4-related
traits is particularly well represented in different species of
the genus Flaveria. In addition to species with typical C3 or
C4 photosynthesis, this genus also includes up to 10 intermediate species (Lyu et al. 2015). Detailed phylogenetic analyses
showed that C3–C4 intermediacy evolved more than once in
the genus Flaveria and that the C3–C4 intermediates represent
starting points for the evolution of C4-like and true C4 photosynthesis (McKown et al. 2005, Lyu et al 2015). This made
Flaveria a preferred research object for the investigation of
physiological, biochemical and phenotypic adaptation during
evolution from C3 to C4 photosynthesis (Ku et al. 1983, Monson
et al. 1986, McKown and Dengler 2007, Mallmann et al. 2014,
Lyu et al. 2015). Hence the current model of the evolution of C4
photosynthesis is based to a large extent on work in C3, C3–C4
and C4 Flaveria species (Monson and Rawsthorne 2000, Sage
2004).
Computational Modeling of the Evolution of
C4 Photosynthesis from C3 Ancestors
Recently, new insights into the evolution of C4 photosynthesis
came from the application of different modeling approaches.
This work capitalized on a mechanistic biochemical model of
photosynthetic CO2 assimilation that was previously developed
by von Caemmerer (1989, 2000). Using the well-characterized
kinetic constants of RuBisCO and various gas exchange characteristics, this model accurately predicts the amount of CO2
assimilated per unit leaf area and time, given a defined CO2
concentration and amount of RuBisCO. The conceptual advance by Heckmann and colleagues (2013) was that they
used this mechanistic model to simulate a fitness landscape
for the transition from C3 to C4, defining the overall fitness
gain as proportional to the amount of CO2 that can be fixed
using a given quantity of RuBisCO per leaf area. In other words,
Heckmann et al. (2013) assumed that higher assimilation rates
with a given amount of RuBisCO, light and water equals higher
fitness since more biomass can be gained with identical input of
resources. The simulation included the following parameters: x,
the fraction of photorespiratory glycine moved for decarboxylation from M to BS cells; b, the fraction of RuBisCO active sites
in M and BS; Vpmax, the activity of the C4 cycle expressed as
maximal velocity of the committing enzymatic reaction, PEPC;
Kccat, the maximal turnover rate of RuBisCO; gs, the BS gas
conductance; and Kp, the Michaelis–Menten constant of
PEPC for its substrate phosphoenolpyruvate (PEP). These six
parameters were varied systematically in computational
884
simulations, and the resulting CO2 assimilation rates calculated
using the mechanistic model, which resulted in a six-dimensional fitness landscape. Two main conclusions could be drawn
from the model: first, the computational model predicts a
modular sequence of events that is consistent with the succession of events predicted by the experimental studies (Monson
and Rawsthorne 2000, Sage 2004, Leegood 2013). In agreement
with previous phylogenetic analyses, it further supports the
hypothesis that the identified C3–C4 intermediates indeed represent transitory stages on the path to C4. Importantly, the
computational simulation predicts that in anatomically preconditioned plants the first step towards C4 photosynthesis is
indeed the relocation of GDC activity from M to BS cells.
Secondly, the path through the biochemical fitness landscape
is smooth, without local minima or maxima, because there
always exists at least one possible parameter change towards
C4 that is associated with immediate fitness gain under the
given environmental conditions.
The evolution of C4 photosynthesis is therefore predicted to
be repeatable in different species and provides a convincing
explanation for the phylogenetic diversity of C4 species. The
robustness of C4 evolution in different phylogenetic backgrounds was tested in a Bayesian modeling approach by
Williams and colleagues (2013). Their study includes 37 species
from 22 genera, probably representing 18 distinct evolutionary
origins of C3–C4 intermediacy. Their model confirmed the predicted sequence of key events on the evolutionary path to C4,
but it also showed that considerable flexibility exists for the
shaping and succession of adjusting steps concerning biochemistry as well as parallel changes in cellular structure and leaf
anatomy. The flexibility of the various biochemical, cell biological and anatomical parameters probably contributes to the
facilitation of convergent evolution of the complex trait. In
agreement with the phylogenetic and biochemical models,
the Bayesian approach also places the described C3–C4 intermediates on the evolutionary trajectory from C3 to C4 (Williams
et al. 2013).
Taken together, the computational studies indicate that C4
evolution is characterized by essential key events starting with
the shift of GDC activity to the BS followed by a flexible succession of adjustment steps. The most recent findings on mechanistic realization of the key events on the path to C4 are
summarized in the next paragraphs.
Pre-Conditioning for C4 Photosynthesis
Despite the predictable modular trajectory of C4 evolution presented above, C4 photosynthesis is still only present in approximately 3% of the angiosperms (Osborne and Sack 2012, R.F.
Sage et al. 2011). Apparently several pre-conditions must be
met before traveling the road to C4 evolution can be initiated.
Environmental conditions have, of course, a major impact on
these events. As evident from the computational models discussed above, C4 evolution would be promoted under conditions that cause high photorespiratory rates, in particular under
low atmospheric CO2 and limiting water supply, in warm and
Plant Cell Physiol. 57(5): 881–889 (2016) doi:10.1093/pcp/pcw009
open environments (Osborne and Sack 2012). C4 evolution
further depends on certain anatomic and genetic pre-conditions. Within the angiosperms, C4 origins therefore cluster in
some branches while they are absent in others (R.F. Sage et al.
2011). This is particularly obvious in the grasses, where all C4
species identified to date belong to the Panicoideae,
Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and
Danthonioideae (PACMAD) clade and none has been discovered yet in the Bambusoideae, Ehrhartoideae and Pooideae
(BEP) clade, to which many of our most important crop species
belong, such as wheat and rice. Analysis of leaf anatomical traits,
such as vein spacing and size of BS cells, in 157 grass species in
combination with phylogenetic and statistical analysis clearly
associated C4 evolvability with the presence of anatomic enablers, in particular relatively large BS cells and low numbers of
M cells between veins (Christin et al. 2013). These lead to
increased proportions of vascular and BS tissue in relation to
total leaf cross-sectional area (Christin et al. 2013). On the biochemical level, shifting GDC activity from M to the BS cells has
been identified as a decisive initial event on the road to C4, but
the successful implementation of a photorespiratory glycine
shuttle obviously depends on a proper anatomic set-up of
the leaf, given the need for transport of photorespiratory glycine to a specific cell type in the leaf. That is, a low M to BS cell
ratio is expected to promote the installation of a glycine-based
photorespiratory carbon pump (Sage et al. 2014).
The genetic make-up of a particular plant species also either
constrains or promotes C4 evolvability (Williams et al. 2012). C4
biochemistry depends on enzyme activities that are present in
all C3 species, but during evolution of C4 become recruited into
a new metabolic context, primarily by changes in enzyme abundance and cell specificity of enzyme expression. The presence of
multiple copies of genes encoding these enzymes (and other
factors required for C4) is therefore likely to be advantageous, so
that one copy of the gene will continuously support the C3
housekeeping function whereas the second copy can be recruited to C4 metabolism without jeopardizing vital functions
associated with the C3 ortholog. The prominent role of gene
duplications as a genetic enabler of C4 evolution has recently
been demonstrated as a prerequisite for the shift of GDC activity from the M to the BS in Flaveria (see below; Wiludda et al.
2012, Schulze et al. 2013).
Installation of a Photorespiratory Pump by
Shift of GDC Activity to BS Cells: An Early and
Essential Step on the Road to C4
According to our current knowledge, the number of C4 lineages
and species on Earth by far outnumbers that of C3–C4 intermediates. R.F. Sage et al. (2011) estimate that we know about
7,500 C4 species, but they list only 43 intermediates. Because
intermediates are slightly more difficult to detect (requiring gas
exchange analysis), it is possible that the proportions presented
above do not accurately mirror the true picture, but the currently available numbers and the proximity of the majority of
C3–C4 intermediates to C4 species indicate that after the
essential initial step is done, the likelihood of full C4 evolution
increases significantly. This view is also supported by the computational models of C4 evolution discussed above (McKown
et al. 2005, Heckmann et al. 2013, Williams et al. 2013,
Mallmann et al. 2014). A detailed mechanistic understanding
of the molecular events that enable the shift of GDC activity
from M to BS cells is therefore of particular importance for
understanding of the entire process, but also for evolutionary-inspired engineering approaches (Denton et al. 2013).
Given the complexity of the GDC system, modification of
GDC activity by random mutations has a relatively high probability. The GDC multienzyme system consists of four different
subunits, and manipulation of any of them has the potential to
change the activity of the entire complex (Engel et al. 2007).
Further targets of random mutations could be any entity that
exhibits regulatory power over any of the subunits (Heckmann
et al. 2013).
In the C3 Flaveria species F. pringlei and F. robusta, two
copies of the GLDP subunit gene exist, GLDPA and GLDPB.
GLDPA is already expressed in a BS-specific manner in C3
Flaveria species (Wiludda et al. 2012), while GLDPB is expressed
in all leaf cells (Schulze et al. 2013). In the C3–C4 intermediate
Flaveria species, the expression of the ubiquitously expressed
GLDPB gene is down-regulated as a consequence of an altered
promoter activity, which causes a shift of GDC activity towards
BS cells because now the GLDPA gene is almost exclusively
responsible for expressing the GLDP subunit of the GDC
system. This disappearance of GLDPB expression from M cells
proceeds gradually from C3 to C4 rather than abruptly, a factor
that is perhaps crucial for the adjustment of the intercellular
metabolism (Schulze et al. 2013). Later on in the evolutionary
trajectory towards C4 in Flaveria, GLDPB gene expression is lost
by pseudogenization, which leaves only the BS-specific GLDPA
gene active. Surprisingly, GLDP expression is not completely
lost from M cells in fully evolved C4. This is due to low expression levels of GLDPA in M cells, which is the result of less stringent BS-specific expression of the C4 GLDPA promoter
(Wiludda et al. 2012, Schulze et al. 2013). Apparently, low
levels of active GDC in M cells are also essential for maintenance of basic cellular functions in the C4 species (Schulze et al.
2013). The presence of a BS-specific GLDP gene already in C3
Flaveria species most probably is a powerful enabler for C4
evolution in this genus. For comparison, the Arabidopsis
genome harbors two GLDP genes with apparently redundant
functions, which allows compensation when just one copy is
lost (Engel et al. 2007). The evolutionary scenario in Flaveria is
probably lineage specific and it will hence be required to unravel the mechanisms underlying cell-specific GDC expression
in independent cases of C4 evolution in other phylogenetic
groups.
The Photorespiratory Glycine Shuttle is
Mechanistically Linked to C4 Evolution
The implementation of the photorespiratory glycine shuttle (in
some publications also called the C2 shuttle or C2
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U. Schlüter and A. P. M. Weber | The road to C4
photosynthesis) between M and BS cells of the early C3–C4
intermediates causes a metabolic imbalance between the two
cell types. In the initial description of the process, Rawsthorne
et al. (1988b) already pointed out that one of the downstream
products of the glycine decarboxylation reaction would need to
move back to the M for replacement of the carbon lost from
the CBC by oxygenation. The GDC reaction in conjunction with
serine hydroxymethyltransferase (SHMT; Voll et al. 2006) uses
two molecules of glycine for the production of one molecule of
serine. In addition, CO2 and ammonium are released. If we now
assume that serine is the product of the GDC/SHMT reaction
that is transported back to the M cells, continuous operation of
the photorespiratory glycine shuttle would therefore also require re-balancing of N metabolism between the cells because
for two units of N in the form of glycine that are imported into
BS cells only one unit in the form of serine is returned. This
potential N imbalance was already mentioned in the original
report by Rawsthorne et al. (1988b), but until recently did not
receive much attention. Mallmann et al. (2014) applied a metabolic modeling approach to explore potential routes for readjustment of the N balance and to identify candidate
metabolites and enzymes involved in such an N shuttle.
When the model was optimized for maximization of leaf biomass production and minimization of the sum of all fluxes, the
model suggests three alternative shuttle mechanisms: (i) glutamate/2-oxoglutarate; (ii) alanine/pyruvate (Fig. 1B); and (iii)
aspartate/malate. Introduction of even low PEPC activity in the
M cells already leads to the prediction of a partial C4 shuttle
mechanism in the C3–C4 intermediate plants, including the
activity of alanine aminotransferase, glutamate-glyoxylate aminotransferase, pyruvate:phosphate dikinase, NADP-malic
enzyme and malate dehydrogenase. The predictions of the
metabolic model were then experimentally tested using nine
different Flaveria species, representing the full spectrum from
C3 via C3–C4 intermediates to C4. Indeed, the model predictions
could be confirmed, with species with an increasing degree of
C4-ness displaying the predicted changes in the mRNA and
protein amounts of the corresponding enzymes. These results
imply that the establishment of the photorespiratory glycine
shuttle already represents a strong enabler for C4 photosynthesis because the shuttle requires ‘C4’ enzyme activities in support
of re-establishing the N balance between M and BS cells
(Mallmann et al. 2014). At this point, these findings cannot
yet be generalized; additional studies in different phylogenetic
groups displaying independent origins of C3–C4 intermediate
photosynthesis will be needed to test the generality of the
presented evolutionary model.
It is interesting to note, though, that in C3 plants PEPC activity is regulated by a variety of metabolic intermediates, in
particular by the glutamine to glutamate ratio, which serves as
an indicator of the N status of the plant, and by ammonium,
both causing increased activity of PEPC (for a review, see Britto
and Kronzucker 2005). Possibly the massive export of glycinebound N from the M to the BS and its release in the form of
ammonium by GDC leads to an N surplus signal in BS cells that
triggers the anaplerotic production of organic acids by PEPC as
backbones for ammonia assimilation in the M. An alternative
886
solution for balancing N metabolism between the M and BS
would be a relocation of the peroxisomal steps of photorespiration to the BS, which would result in glycolate import and
glycerate export, without any net N movement (i.e. a glycolate/
glycerate instead of a glycine/serine shuttle). Interestingly, in C4
plants, such as corn, all steps of photorespiration with the exception of the final one, glycerate kinase, are located within the
BS (Baldy and Cavalié 1984). Apparently, in C3–C4 intermediate
plants that display high RuBisCO activity in the M, the high
concentrations of glycolate that would be required for driving
its rapid diffusion from the M to the BS is not tolerated, whereas
relatively high glycine concentrations are seemingly permitted.
Physiology of C3–C4 Intermediates
In contrast to the now well-developed biochemical C3–C4
model, information on the physiology of C3–C4 intermediates
is still limited. A first model of gas exchange in C3–C4 intermediates was presented by von Caemmerer (1989). It demonstrated the quantitative feasibility of this pathway and provided
a mathematical explanation for C3–C4 characteristic features
such as the reduction of the CO2 compensation point by operation of the glycine shuttle (Fig. 1B).
Measurements of CO2 compensation points by infrared gas
exchange analysis are, however, influenced by multiple factors
and they do not permit the consideration of leaf internal differences (Keerberg et al. 2014). Dynamic analysis of 14CO2 release from labeled photosynthates generated by long-term
exposure of leaves to 14CO2 recently enabled an estimation of
the different fluxes within the leaf. Comparison of Flaveria pubescens, a C3–C4 intermediate species, with its C3 relative
F. cronquistii indicates that the photorespiratory pump elevates
the mean intraplastidial CO2 concentration during steady-state
photosynthesis by about 3-fold (Keerberg et al. 2014). In an
ecological context, this would translate into a fitness advantage
under C-limiting conditions.
In contrast to the situation in C3 and C4 plants, the CO2
compensation point of C3–C4 intermediates is strongly influenced by environmental conditions, especially CO2, temperature and light (Brown and Morgan 1980, Holaday et al. 1982,
Hunt et al. 1987). Under low light conditions, the CO2 compensation point of C3–C4 intermediates is almost C3-like but
then it declines under increasing illumination. Hence the efficiency of the glycine pump is dependent on high rates of ribulose 1,5-bisphosphate regeneration and glycine production
(Sage et al. 2013). Compared with C3 relatives, C3–C4 intermediates from the genera Flaveria, Heliotropium and Alternanthera
showed superior photosynthetic performance, especially under
high temperature and low CO2 concentrations (Vogan and
Sage 2012). This correlates with the predicted evolution of
C3–C4 intermediates and C4 ancestors in open habitats under
conditions of low CO2 and high temperatures (Osborne and
Sack 2012).
Fully evolved C4 plants also possess higher water (WUE) and
nitrogen use efficiency (NUE) than C3 plants. Due to their efficient carbon concentration mechanism, C4 plants display high
Plant Cell Physiol. 57(5): 881–889 (2016) doi:10.1093/pcp/pcw009
assimilatory rates even when stomatal conductance is low. The
C4 carbon pump allows for a reduced investment into RuBisCO
protein and hence N, which translates into increased rates of
assimilation of carbon per unit leaf nitrogen. For C3–C4 intermediates, the picture is not consistent. In Heliotropium species,
the reduction in the CO2 compensation point was associated
with enhanced WUE (Vogan et al. 2007). Assessment of WUE
and NUE in Flaveria species with different degree of C4-ness
could, however, not confirm a positive effect on WUE and NUE
of the early intermediates (Kocacinar and Sage 2008, Vogan and
Sage 2011, Way et al. 2014). Only at the later stages of C4 evolution, when a C4 pump is already in operation, did the water
economy improve significantly (Kocacinar and Sage 2008, Way
et al. 2014). Similar results were obtained for the monocotyledonous C3–C4 intermediate Panicum milioides (Pinto et al.
2011).
Advantages of the intermediate photosynthesis type seem to
occur mainly under conditions of high temperature and the low
CO2 conditions present in recent geological times (Vogan and
Sage 2012). Under specific environmental conditions, plants
could, however, also benefit from intermediate photosynthesis
under present CO2 conditions. In Flaveria, C3–C4 intermediate
species tended to have shorter vessels than C3 relatives, which
could contribute to xylem safety under very dry conditions
(Kocacinar and Sage 2008). Carbon economy of intermediates
could also be advantageous when low stomatal conductance
limits CO2 concentrations within the leaf. A general influence
of the photorespiratory pump on whole-plant water economy,
on the other hand, could not be established to date.
Outlook on Impact of New Technologies on
Studies of C4 Evolution
In recent years, the study of C3–C4 intermediates, mainly from
the Flaveria genus, contributed substantially to the development
of advanced models explaining the biochemical aspects of the
evolution of C4 photosynthesis. These models suggest that after
the initial installation of a photorespiratory glycine pump, implemented by altered cell specificity of GDC expression, evolution
can continue towards C4 on a smooth path through a Mount
Fuji-like fitness landscape (Heckmann et al. 2013, Mallmann et al.
2014). Changes in metabolic capacities on this path seem to be
gradual rather than discrete steps, and consist of a multitude of
metabolic adjustments (Heckmann et al. 2013). The succession
and shaping of these adjustments seem to be very flexible and
robust in different genetic backgrounds (Williams et al. 2013). So
far the detailed studies in the genus Flaveria confirmed the
events predicted by the models, and it will be interesting to
compare them with scenarios from other C4 lineages. Over the
course of the next few years, the study of C3–C4 intermediates
will be particularly exciting in the following fields.
Application of transcriptome and
genome sequencing
With the advent of next-generation sequencing, the cost of
transcript and genome sequencing continues to drop
dramatically while throughput is constantly increasing
(Weber 2015). This provides exciting new opportunities for
the investigation of C4 evolution. As demonstrated for the
case of GLDP in Flaveria, detailed genetic information is crucial
for elucidating the mechanisms underlying some of the key
events of C4 evolution. Investigation of phylogenetically distant
systems will increase the power to understand potential plasticity within the evolutionary trajectory towards C4. New species with C3–C4 intermediate photosynthesis are continually
being discovered. In a detailed review by Sage et al. (2012), 21
different intermediate lineages have been described. The majority of those have close C4 relatives, but for only nine of them
no progression towards C4 could be demonstrated so far, and
among them are also at least two lineages from the Brassicales,
for example Moricandia species and Diplotaxis tenuifolia. There
are reports that in the genus Alloteropsis, C3 and C4 photosynthesis are even found within the same species (Lundgren et al.
2015). Construction of advanced high-resolution phylogenies
will aid in the design of new experimental systems for identification of additional C4-specific parameters. So far, only genes
involved in biochemistry of the trait are well described, but
additional players such as regulatory elements and factors influencing organelle, cell and leaf structure are still mostly unknown. Besides the biochemically relevant genes, these could
also have great influence on the advancement or retardation or
even abortion of C4 evolution.
Dynamic flux analysis
The work of Keerberg et al. (2014) clearly showed that dynamic
metabolic flux analysis is essential to dissect fully the events in
the different leaf compartments. Such knowledge will be
required for the identification of events that exert the greatest
instant effect on photosynthesis of the leaf. Dynamic flux experiments with 13C helped recently to advance our knowledge
on photosynthetic events in Arabidopsis leaves (Szecowka et al.
2013, Ma et al. 2014). Application of similar methods to C3–C4
intermediates will promote a more detailed and mechanistic
understanding of the evolutionary drivers beside the
biochemical networks. Knowledge of the influence of
environmental factors on C3–C4 photosynthesis is also very
limited. Particularly interesting will be studies on the
interplay of light, CO2 and water of the C3–C4 photosynthetic
dynamic.
Quantitative genetics analysis of hybrids between
C3 and C3–C4
For a number of C3–C4 intermediates genetic crosses to related
C3 species that produce a fertile hybrid offspring have been
reported. Successful crossings have been performed between
Flaveria species with C3- and C4-like photosynthesis (Holaday
et al. 1988). Moricandia arvensis (C3–C4) can be hybridized with
the crop species B. napus and B. oleracea (Apel et al. 1984,
McVetty et al. 1989. Bang et al. 2009). Also M. moricandioides
and M. arvensis can be hybridized, which enables genetic
approaches towards identifying the genes controlling C3–C4
intermediate photosynthesis. In combination with genome
sequencing and deep genotyping by next-generation
887
U. Schlüter and A. P. M. Weber | The road to C4
sequencing, the analysis of segregating populations of these
hybrids provides a powerful tool to identify genes that
encode enablers of C4 evolution. This knowledge can then be
translated into breeding programs that aim at increasing
photosynthetic energy conversion efficiency.
Summary
The recent application of different modeling approaches allowed a
refinement of the current model of C4 evolution via C3–C4 intermediate stages. The models predict that the evolution from C3 to
C4 is characterized by a succession of common key events starting
with the implementation of the photorespiratory glycine pump
and the susequent need for additional balancing steps between
the M and BS. A multitude of additive adjustment steps seems to
be flexible in sequence and shape, with continuous fitness gains on
the path towards C4 photosynthesis (Heckmann et al. 2013,
Williams et al. 2013, Mallmann et al. 2014). Plant species with
features between C3 and C4 have been confirmed as true intermediate stages on the trajectory to C4, independently by phylogenetic as well as biochemical modeling. The modeling work is also
in agreement with the detailed experimental work done
(Heckmann et al. 2013, Williams et al. 2013, Mallmann et al.
2014) especially in Flaveria species on very different stages of C4
evolution. In the future it will be interesting to see if lack of progression to C4 in some lineages is likely to be connected to chance
and the absence of selective pressure or if specific anatomical and
metabolic changes can be identified which prevented progression
towards C4.
Funding
Work on the evolution and function of C4 photosynthesis is
suppoted by the Deutsche Forschungsgemeinschaft [grants
WE2231/8-2, WE2231/9-2, IRTG 1525 and EXC 1028].
Acknowledgements
We thank the anonymous reviewers for their constructive suggestions and input on this manuscript.
Disclosures
The authors have no conflicts of interest to declare.
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