Journal of Experimental Botany, Vol. 68, No. 2 pp. 97–102, 2017
doi:10.1093/jxb/erw491 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
SPECIAL ISSUE EDITORIAL
C4 photosynthesis: 50 years of discovery and innovation
It is now over half a century since the biochemical characterization of the C4 photosynthetic pathway, and this
special issue highlights the sheer breadth of current
knowledge. New genomic and transcriptomic information shows that multi-level regulation of gene expression
is required for the pathway to function, yet we know it to
be one of the most dynamic examples of convergent evolution. Now, a focus on the molecular transition from C3–
C4 intermediates, together with improved mathematical
models, experimental tools and transformation systems,
holds great promise for improving C4 photosynthesis in
crops.
The year 2016 marked 50 years since the first published biochemical characterization of the C4 photosynthetic pathway
by Hal Hatch and Roger Slack (Box 1; Hatch and Slack,
1966). Following the experimental designs of Calvin and coworkers, they used 14CO2 to trace the fate of CO2 assimilated
by sugarcane and confirmed that the first carbon compound
formed was a C4 acid. This led to the definition of the C4
dicarboxylic acid pathway, later abbreviated to C4 photosynthesis, and the plants employing this process were termed
C4 plants. Furbank, in one of two comprehensive Darwin
reviews in this issue, retraces these historical events in detail
(Furbank, 2016). After the seminal experiments by Hatch and
Slack, unravelling the biochemistry of the pathway in a number of species followed rapidly and provided the foundation
of our current knowledge on the diverse biochemistry of C4
photosynthesis (Hatch, 1987; Hatch, 1992; Furbank, 2016).
The C4 pathway isn’t just about biochemistry, rather it is a
complex combination of biochemical and morphological specialization. Most C4 species are characterized by the so-called
Kranz anatomy, with Rubisco located in specialized cells
adjacent to the vascular tissue (bundle sheath cells) and PEP
carboxylase in the mesophyll cells. It is the gas-tight nature of
the bundle sheath that allows the decarboxylation of C4 acids
Box 1. Pioneers of C4 photosynthesis
Roger Slack, Hilary Warren and Hal Hatch at the opening of the conference ‘C4 Photosynthesis:
past, present and future’ in April 2016. The meeting was held at the ARC Centre of Excellence
for Translational Photosynthesis in Canberra, Australia to commemorate the discovery of the
C4 pathway and its significance in today’s plant biology and agricultural research (see Hibberd
and Furbank, 2016). Hilary Warren (then Johnson) was the first PhD student of Hal Hatch. It is
with sadness that we note that Roger Slack passed away on 24 October 2016.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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.
98 | Editorial
in this compartment to elevate CO2 partial pressure around
Rubisco. This inhibits its oxygenase activity allowing it to
operate close to its maximal rate.
Despite this complexity, C4 photosynthesis is recognized as
one of the most dynamic examples of convergent evolution,
arising multiple times over the last 60 million years in warm
semi-arid regions, with early occurrences coinciding with low
atmospheric CO2 in the late Oligocene (Sage et al., 2011; Sage,
2016). In his Darwin review, Sage (2016) outlines the evolution of the 61 independent C4 lineages which have resulted in
more than 8000 species in grasses, sedges and eudicots and
looks at the biogeography of these species.
C4 plants play a key role in world agriculture – crops such
as maize and sorghum are major contributors to world food
production in both developed and developing nations, and
the C4 grasses sugarcane, miscanthus and switchgrass are the
major plant sources of bioenergy. In comparison to C3 crops
such as rice, C4 crops have higher yields and increased water
and nitrogen use efficiency (Hibberd et al., 2008; Langdale,
2011). The agronomic use of C4 species, as well as their
substantial influence on terrestrial CO2 fixation (Still et al.,
2003), provides the scientific drive for understanding what
has allowed the evolution of C4 photosynthesis to happen so
many times.
This special issue follows two other recent volumes of Journal of Experimental Biology focused on C4
(‘Exploiting the engine of C4 photosynthesis’ – Volume 62,
Issue 9, see Sage and Zhu, 2011; and ‘C4 and CAM photosynthesis in the new millennium’– Volume 65, Issue 13, see Sage,
2014). The papers here continue the C4 story and highlight
the diversity of current research in the quest to get a better
understanding of the C4 photosynthetic process and enable
crop scientists to perhaps imitate the process of C4 evolution
and turn C3 plants into C4 plants (Box 2).
What can we learn from genomes and
gene regulation in C4 species?
Furbank (2016) points to a wealth of genomic and transcriptomic information now available for C4 leaves, and leaves of
closely related C3 plants, which is catalysing a new generation
of research into the C4 mechanism and the genetic architecture underpinning it. A transcriptomics/genomics approach
and a review of gene expression across multiple lineages of
C4 plants (Aubry et al., 2014; Williams et al., 2016; Reeves
et al., 2017) have led to the conclusion that regulation of
gene expression at multiple levels (including transcriptional
control by promoter regions, as demonstrated by Gowik
et al., 2017) is required for C4 photosynthesis to function. It
appears that posttranscriptional control may also be important (Fankhauser and Aubry, 2017) and that many of the
Box 2. The multidisciplinary approaches used and needed to unravel the secrets of C4
photosynthesis
The C4 photosynthesis conference in Canberra in 2016 brought together world experts in the
field ranging in discipline across biochemistry, physiology, molecular genetics and ecophysiology, and also included those involved in applied efforts to engineer C4 into C3 crops. Currently,
a renewed research focus on C3–C4 intermediate species is unearthing more intermediate species and new evidence for the molecular transition from the C3 to the C4 state. The description
of improved mathematical models, combined gas exchange and stable isotope tools, metabolic 13CO2 labelling kinetics and more efficient transformation systems for C4 plants (such as
Setaria viridis) hold great promise for improving C4 photosynthesis in a crop environment.
Editorial | 99
mechanisms for regulation of C4 gene expression are indeed
present in C3 plants and recruited to a C4 function (Reeves
et al., 2017). Denton et al. (2017) remind us, though, that caution must be used in interpreting gene expression data, particularly cell- or tissue-specific data, which may include biases
due to RNA preparation methods. The utility of comparative
genomics in this field is shown by Huang et al. (2017), who
have developed a cross-species genome scanning approach to
identify genes under positive selection in C4 evolution which
is independent from knowledge of the biochemical pathways
involved (see also the Insight article in this issue by Christin,
2017). It is interesting that molecular genetics, genomics
and transcriptomics are now commonly being used in a biochemical and evolutionary research perspective as affordable approaches to answer questions in C4 photosynthesis
research, rather than operating in isolation as stand-alone
fields (Box 2).
New insights from phylogeny and C3–C4
intermediate species
Since their discovery, C3–C4 intermediate species have been
hypothesized to be evolutionary intermediates on the path
to or from C4 photosynthesis (Peisker, 1986; Monson and
Moore, 1989; Sage et al., 2012; Heckmann et al., 2013).
Biochemical and molecular studies have elucidated the various types of photosynthetic intermediacy, which range from
the simpler C2 mode involving the glycine or photorespiratory
shuttle with rudimentary bundle sheath to a C4-like pathway
with well-developed Kranz anatomy and functional C4 pathway (Sage et al., 2012). Physiological studies have revealed a
clear lowering of the CO2 compensation point (CO2 partial
pressure where there is no net CO2 exchange) for all types
of C3–C4 intermediates, but advantages related to improved
water and nitrogen use efficiency are only expressed in intermediate plants possessing a degree of C4 acid fixation (Vogan
et al., 2011; Pinto et al., 2016). More recent studies, including
those represented in this issue, have focused on documenting the phylogenetic diversity of C3–C4 taxa and elucidating
the molecular elements underscoring the evolutionary, and
in rare cases, the developmental, transitions from C3 to C4
(Gowik et al., 2011). The prized goal has been the mining of
C3–C4 species to identify anatomical, biochemical and molecular features that underlie C4 evolution.
In this issue, both Voznesenskaya et al. (2017) and Schüßler
et al. (2017) report on phylogenetic searches for C3–C4 species. Voznesenskaya et al. (2017) demonstrate that the family
Portulacaceae has a C3–C4 Cryptopetala clade and a diverse
C4 Pilosa clade, while Schüßler et al. (2016) resolve the
C3–C4 intermediate and C4 lineages in the Salsoleae family
(Chenopodiaceae). Lauterbach et al. (2017) go a step further
and combine physiological, anatomical and transcriptomic
approaches to elucidate the molecular transition from the C3
to the C4 state in the leaves of Salsola soda (Chenopodiaceae).
Kümpers et al. (2017) use leaf maturation in C3 and C4 Flaveria
species to identify transcription factors. Schlüter et al. (2017)
draw our attention to limitations connected to N metabolism
and vein density that may have constrained the evolutionary transition of two Moricandia species (Brassicaceae) from
C3–C4 into the C4 pathway. Regardless of phylogenetic constraints, Lundgren and Christin (2017) demonstrate that the
evolution of the C3–C4 pathway brings intermediate species
into C4-like environments facilitating C4 evolution.
New technologies and mathematical
models elucidate the physiology and
biochemistry of C4 photosynthesis
Early discoveries of C4 photosynthesis made use of new
physiological techniques such as gas exchange measurements.
This led to the development of distinguishing gas exchange
features of C4 CO2 assimilation rates. We now have a good
understanding of how C4 photosynthesis responds to environmental variables such as light, temperature and CO2 (Long,
1999). The first biochemical model of C4 photosynthetic gas
exchange correctly predicted its CO2 concentrating function,
with first estimates of bundle sheath CO2 partial pressures,
although we still don’t know what they actually are (Berry and
Farquhar, 1978). These functional models of C4 which allow
the link between leaf biochemistry and gas exchange have
become essential tools (von Caemmerer and Furbank, 1999;
von Caemmerer, 2000). Bellasio (2017) has combined these
models to generate a general stoichiometric model for C3, C2,
C2+C4, and C4 photosynthesis in which energetics, metabolite
traffic and the different decarboxylating enzymes are explicitly
included. The model comes with an Excel spreadsheet inviting the community to have a go at redesigning C4 photosynthesis. The usefulness of a sound mathematical framework is
also highlighted in the opinion paper by Li et al. (2017), who
use these models (following Heckmann et al., 2013) to outline how to combine genetic and evolutionary engineering to
establish C4 metabolism in C3 plants.
Most C4 species are characterized by Kranz anatomy, but
there are a small number, such as Bienertia cycloptera, that
perform C4 photosynthesis within individual mesophyll cells
(Voznesenskaya et al., 2001; King et al., 2012). By modelling the processes of diffusion, capture and release of CO2
and oxygen inside a typical Bienertia mesophyll cell, Jurić
et al. (2017) show that a spatial separation as low as 10 μm
between the primary and the secondary carboxylases can
provide enough diffusive resistance to sustain an efficient C4
pathway, demonstrating that single-cell C4 photosynthesis
is a viable option. CO2 diffusion during C4 photosynthesis
also remains an important issue in those species with Kranz
anatomy. Theories developed for the interpretation of stable
isotope discrimination during C4 photosynthesis (Farquhar,
1983; Gillon and Yakir, 2000; Barbour et al., 2016) allow us
to probe the interconnectivity of C3 and C4 cycle activity and
CO2 diffusion properties into mesophyll cells. For example,
13
CO2 isotope discrimination can be used to quantify bundle sheath leakiness (the ratio of CO2 leak rate out of the
bundle sheath over the rate of CO2 supply) and C18OO discrimination allows quantification of CO2 diffusion from
intercellular airspace to the mesophyll cytosol in relation to
100 | Editorial
carbonic anhydrase activity there. Recent technical advances
have greatly facilitated the measurements of isotope discrimination concurrently with gas exchange (Gong et al.,
2017; Osborn et al., 2017). Gong et al. (2017) documented
dynamic variation in bundle sheath leakiness of a perennial
C4 grass with short-term variation in atmospheric CO2 concentration. Osborne et al. (2017) generated transgenic Setaria
viridis plants with reduced carbonic anhydrase activity and
used measurements of C18OO discrimination to show that
carbonic anhydrase and mesophyll conductance are both
limiting factors affecting CO2 assimilation rates at low CO2
partial pressures.
The techniques of the 14C pulse chase which were used by
Hatch and Slack to unravel the mysteries of C4 photosynthesis
have been replaced by mass spectrometric measurements of 13CO2
labelling kinetics, which provide a wealth of information compared to past experiments. This technique was used for the first
time by Arrivault et al. (2017) in maize to establish pool sizes and
gradients of metabolites using cell type fractionation. It will provide a welcome tool for establishing C4 metabolism in C3 species.
Today, major C4 crops are grown in dense stands where
most leaves are shaded compared to their wild progenitors.
Pignon et al. (2017) show that leaves of two highly productive
C4 crops lose photosynthetic efficiency in low light as they
become shaded by new leaves, costing the crop up to 10% of
its yield potential. The ancestors of maize and miscanthus
appear to have existed in very open habitats, where water
and nutrient deficiencies would have limited leaf area. There
may therefore have been little evolutionary pressure for maintenance of photosynthetic efficiency in shade conditions.
Improving C4 photosynthesis in a crop environment may be
an important next step for increasing genetic yield potential
in some of these most important crops (Long et al., 2015; von
Caemmerer and Furbank, 2016).
Future perspectives
Where will C4 research go next? In 50 years we have seen the
expansion of the field from the examination of a rudimentary biochemical pathway in just a few species to the construction of complex evolutionary models and assembly of
massive genomic and transcriptomic data sets from a large
range of both crop and wild C4 species, as well as multiple
efforts to engineer C4 traits into C3 crops and model species.
As gene and transcript sequencing costs plummet with thirdgeneration technologies, what will be the new technological
driver of C4 research?
From a biochemical and modelling perspective, the confounding nature of the two-compartment C4 system for ‘grind
and find’ extraction of metabolites, transcripts and proteins
has been a challenge. Recently, however, high resolution
MALDI imaging mass spectrometry was used to examine
the lipid composition of thylakoids of mesophyll and bundle
sheath cells of maize (Dueñas et al., 2016). With appropriate rapid kill and cryopreservation, this technique may hold
promise for measuring metabolites during photosynthesis in
mesophyll and bundle sheath compartments more accurately.
Gene discovery through genomics approaches reveals
gene candidates and evidence for the importance of certain
genes in evolution or for plant performance, but these must
be experimentally validated. Commonly, for C3 dicots, this
is done in model systems like Arabidopsis or tobacco by
gene inactivation or overexpression, but only recently have
grass transformation systems become sufficiently routine for
researchers to approach these experiments in their laboratories. Alternatives such as producing large panels of mutants
by non-targeted mutagenic approaches or by crossing genetic
material to develop near-isogenic lines with and without
genetic polymorphisms is outside the scope of most small
research laboratories. Future development of new and more
efficient transformation systems for a range of C4 plants and
the development of genetic stocks which can be ordered routinely for knockout lines and backcrossed mutants, sequenced
populations and recombinant in-bred lines would see a
rapid development in C4 research similar to that seen when
Arabidopsis genetic resources became widely available.
In the field of C4 engineering, synthetic biology has the
potential to impact hugely on both basic and basic/strategic engineering approaches (Schwander et al., 2016). In the
case of the C4 rice project, the ability to make multiple gene
constructs simplifies cloning strategies (Simkin et al., 2015).
Similarly, if CRISPR/Cas9 technology is combined with
high-efficiency C4 grass transformation systems, production
of allele mimics of potentially important genes occurring
in nature and engineering of novel enzyme properties in C4
plants would advance rapidly.
There is a vast array of information and new technology
now at our fingertips. Nevertheless, we must still marvel at
the achievements of researchers 50 years ago in assembling a
completely new photosynthetic pathway from a collection of
radiolabelling experiments and enzyme assays, and the rapidity with which these researchers brought C4 anatomical and
biochemical data together to underpin the knowledge of the
C4 mechanism we have today.
Key words: 14C pulse chase, C3–C4 intermediate species, C4 crops,
C4 photosynthetic pathway, C4 rice project, CO2 fixation, convergent
evolution, Hal Hatch and Roger Slack, Kranz anatomy.
Susanne von Caemmerer1,*, Oula Ghannoum2 and
Robert T. Furbank1
1 Australian Research Council Centre of Excellence for
Translational Photosynthesis, Division of Plant Sciences, Research
School of Biology, The Australian National University, Acton, ACT
2601, Australia
2 ARC Centre of Excellence for Translational Photosynthesis
and Hawkesbury Institute for the Environment, Western Sydney
University, Richmond NSW 2753, Australia
* Correspondence: [email protected]
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