- © 2010 American Society of Plant Biologists
C4 PHOTOSYNTHESIS—THE BASICS
C4 photosynthesis is a unique blend of biochemical, anatomical, and gene regulatory characteristics. In the vast majority of C4 plants, i.e. with the exception of single-cell C4 photosynthesis in the Chenopodiaceae, this photosynthetic pathway is the result of the integrated metabolic activities of two distinct, specialized leaf cell types, mesophyll and bundle sheath cells. CO2 is initially fixed in the mesophyll cells by phosphoenolpyruvate carboxylase. The resulting C4 acids diffuse into the bundle sheath cells, where CO2 is liberated by one or several of the decarboxylases NADP-malic enzyme, NAD-malic enzyme, or phosphoenolpyruvate carboxykinase, and channeled into the Calvin-Benson cycle. C4 photosynthesis is therefore essentially a pump that concentrates CO2 at the site of Rubisco in the bundle sheath cells (Kanai and Edwards, 1999). The division of labor between mesophyll and bundle sheath cells involves the compartmentation of enzymes and metabolite transporters of the photosynthetic carbon assimilation pathway, but also of the photosynthetic electron transport complexes and other metabolic pathways. It relies on the differential expression of the corresponding genes (Hibberd and Covshoff, 2010).
As a consequence of the CO2-concentrating mechanism Rubisco becomes saturated with CO2, photorespiration is largely reduced, and Rubisco can achieve its maximal catalytic activity. In addition, C4 plants use water and nitrogen more efficiently than C3 species (Ehleringer and Monson, 1993). Not surprisingly, C4 plants contribute about 25% of total terrestrial photosynthesis, although they account for only 3% of the vascular plants. Entire ecosystems like the warm-climate grasslands or savannas are dominated and shaped by C4 plants of the grass family. The emergence of C4 grasses in the late Miocene and Pleistocene, i.e. 3 to 8 million years ago, and the concomitant displacement of C3 grasses demonstrates the adaptational advantage of the C4 photosynthetic pathway (Edwards et al., 2010).
THE C4 SYNDROME—KRANZ ANATOMY AND VEIN SPACING
Although leaf architecture may vary considerably in the various mono- and dicotyledonous C4 lineages, a wreath-like structure of mesophyll and bundle sheath cells around the vascular bundles (Kranz anatomy) is typical for all C4 plants. Mesophyll cells are always located toward the outer face of the leaf and thus in contact with the intercellular air space, while bundle sheath cells are arranged internally to the mesophyll cells and hence close to the vascular tissues. Mesophyll and bundle sheath cells of C4 species are intimately connected to each other due to the high densities of plasmodesmata (Dengler and Nelson, 1999).
Kranz anatomy is accompanied by a dense vein spacing. The high vein density results in a reduced volume of mesophyll relative to bundle sheath tissue that in C4 grasses may reach a ratio of nearly one to one. The close vein spacing in C4 species appears to be mostly due to changes in the patterning of the minor and not the major veins (Ueno et al., 2006).
C4 PHOTOSYNTHESIS IS OF POLYPHYLETIC ORIGIN AND A QUANTITATIVE TRAIT
C4 photosynthesis is found only in higher plants, but originated more than 40 times independently during angiosperm evolution. Most of the C4 species occur in the grasses (approximately 4,600) and sedges (approximately 1,600), whereas only about 1,600 C4 dicots species are known. They are spread over 15 families. C4 grasses probably evolved in the early Oligocene about 30 million years ago, while C4 dicots appeared later, less than 20 million years ago (Sage, 2004).
The polyphyletic origin of C4 photosynthesis indicates that only relatively small evolutionary changes were required for the establishment of this photosynthetic pathway. Studies with genera containing C3, C4, and C3-C4 intermediate species, e.g. Flaveria, allowed insights into the evolutionary trajectories (Sage, 2004). C4 evolution started with genomes that had acquired redundant genes due to duplications of whole genomes, genome segments, or single genes (Monson, 2003). An increase in vein density and an enhancement and activation of the bundle sheath cell layer resulted in a rudimentary Kranz anatomy. The compartmentation of Gly decarboxylase in the bundle sheath cells was the next step and led to a photorespiratory CO2 pump. An elevated phosphoenolpyruvate carboxylase activity and subsequently an increase in the other C4 cycle enzymes and transporters accompanied by their compartmentalized expression established the C4 cycle between mesophyll and bundle sheath cells.
The stepwise transition from C3 to C4 and the occurrence of C3-C4 intermediate species in today’s flora suggests that each of the steps provided an adaptive advantage. Genetically the C4 syndrome may therefore be best described as a polygenic, quantitative trait. The concept of C4 photosynthesis as being a quantitative trait immediately implies a number of questions that will structure the course of future studies into the biology of this physiological and anatomical syndrome.
What is the genetic architecture of C4 photosynthesis, i.e. how many genes are required to establish this phenotypic syndrome? Are the genes organized into functional units giving rise to distinct subphenotypes? Do these functional units form gene regulatory networks whose component genes are regulated coordinately and hence may be viewed as separate regulatory modules?
THE GENETIC ARCHITECTURE OF C4 PHOTOSYNTHESIS
Searching for a Genetically Tractable Model System
Genetic approaches using model systems are the silver bullet to dissect biological traits into its component genes and understand their functions. Model systems are easily genetically accessible, have short generation times, and a small stature. They can be efficiently and robustly transformed and have a small genome.
Maize (Zea mays) and—less so—sorghum (Sorghum bicolor) have been and are still used extensively for C4 research. Their genomes are known, and at least maize can be transformed with reasonable efficiency. Maize and sorghum are large plants with relatively long generation times that make large-scale mutagenesis experiments a logistical challenge. The genome of maize is large and quasitetraploid, which adds to the difficulties in using this plant as a C4 model species.
The Asteracean genus Flaveria has become a model system for studying the evolutionary trajectories taken from C3 to C4 photosynthesis. The genus is ideally suited for this purpose, because it contains large numbers of C3, C3-C4, and C4 species. A C4 species, Flaveria bidentis, is transformable, although with only moderate to low efficiency. A genome sequence is lacking, and it has not been explored whether F. bidentis is suited for forward and reverse genetic approaches.
Because of its phylogenetic relationship with Arabidopsis (Arabidopsis thaliana) the genus Cleome was proposed as a C4 model system (Brown et al., 2005). Cleome includes both C4 and C3 species that grow and flower under greenhouse conditions with short generation times. The C4 species Cleome gynandra can be transformed (Newell et al., 2010); however, as in Flaveria, its genetics is not developed.
The small C4 grass Setaria viridis, the wild progenitor of foxtail millet (Setaria italica), has been discussed as a model system for bioenergy research (Doust et al., 2009). The Setaria genome is small and currently being sequenced. S. viridis plants are of small stature and self-compatible. They have a short life cycle, and genetic resources are already available. Thus S. viridis could be a perfect genetic model for C4 research; however, a robust and efficient transformation procedure is not yet available.
Mapping of C4 Quantitative Trait Loci
The notion of C4 photosynthesis as a quantitative trait suggests that a quantitative trait loci mapping strategy could be pursued to decipher the C4 genetic framework. Such an approach requires closely related C3 and C4 species that can be crossed to give fertile F1 offspring from which F2 populations segregating for C4 traits can be constructed.
Hybridizations between closely related C3 and C4 species were started in the early 1970s. F2 populations were constructed from F1 hybrids between Atriplex hastata (C3) and Atriplex rosea (C4) and shown to segregate for individual C4 traits. However, the F2 plants were aneuploid and further crosses were therefore not feasible. Within the genus Flaveria crosses between C3 and C3-C4 species were achieved, but crosses between C3 and C4 Flaveria species failed (Brown and Bouton, 1993).
Since segregating populations between C3 and C4 Atriplex species are feasible and the development and application of molecular marker technology and high-throughput genome sequencing in nonmodel species is no longer a hindering factor, the hybridization experiments with Atriplex should be revitalized.
The grasses do not contain closely related C3 and C4 species with the exception of the Panicoid grass Alloteropsis semialata that is divided into two subspecies with C3 (subsp. eckloniana) and C4 photosynthesis (subsp. semialata). However, the subspecies differ in ploidy levels (Ueno and Sentoku, 2006), which interferes with hybrid formation.
Evolutionary Studies
The evolutionary transition from C3 to C4 photosynthesis involved massive quantitative and qualitative changes in gene expression. A comprehensive and comparative analysis of gene expression in closely related C3 and C4 species, e.g. Flaveria, Cleome, and Atriplex, should therefore allow identifying the C4 gene repertoire (Bräutigam et al., 2010). Closely related species have to be compared to minimize that the observed expression differences are due to the phylogenetic distance rather than to the presence or absence of C4 photosynthesis. In nonmodel organisms, transcriptome analysis by using next-generation sequencing technologies is the method of choice and may also lay the foundation for proteome approaches.
We believe that Flaveria is the genus of choice in which such a comparative transcriptome analysis should be performed. The genus contains the largest number of C3-C4 intermediate species reported so far in any genus. All the physiological, biochemical, and anatomical characteristics relevant to C4 evolution have been investigated and the data are documented in quantitative formats (compare with McKown and Dengler, 2007). A statistical comparison of transcriptome and phenotypic data should allow detecting correlations between genes and C4 traits.
Using C3 Model Plants for Identifying Genetic Determinants for Kranz Anatomy
Anatomical and molecular biological studies indicate that C3 plants possess already a blueprint for a basic Kranz anatomy (compare with Engelmann et al., 2008). Many C3 angiosperms, like Arabidopsis, have a parenchymateous and even chlorenchymateous bundle sheath. However, in contrast to C4 species this bundle sheath is typically not well pronounced and it contains much less and smaller chloroplasts than in C4 plants (Leegood, 2008).
This finding opens up the possibility to search for Kranz anatomy genes in C3 model species and take advantage of the rich genetic and molecular resources that are available for these species. Understanding the formation of bundle sheath cells and of veins in these C3 species would allow to propose functional models that could be tested in C4 species.
GENE REGULATION IN C4 PHOTOSYNTHESIS
A Case for Gene Regulatory Networks
The evolution of C4 photosynthesis required massive quantitative and spatial changes in gene expression (Bräutigam et al., 2010). It is believed that most of these changes were achieved by transcriptional regulation. However, posttranscriptional control of gene expression in C4 photosynthesis is well documented, and the role of this regulatory level in C4 gene expression may be underestimated (Hibberd and Covshoff, 2010).
How can one reconcile the polyphyletic origin of C4 photosynthesis and the massive changes in gene expression that accompanied each C3-to-C4 transition? All available evidence indicates that preexisting gene regulatory networks in C3 angiosperms were decisive preadaptations that paved the way toward multiple C4 evolution. Promoters driving mesophyll or bundle sheath specific gene expression in C4 species partly maintain their cell preference of expression in closely or even widely related C3 species (Matsuoka et al., 1994; Engelmann et al., 2008). This suggests that the gene regulatory networks controlling the development and differentiation of mesophyll and bundle sheath cells of C4 plants are not fundamentally different from those of C3 species. In addition, gene regulatory networks exist in C3 plants that assure a coordinated response of genes involved in photosynthesis and related metabolic pathways (Mentzen and Wurtele, 2008). As a consequence, networks for regulating both developmental and metabolic processes operated already in C3 ancestral angiosperms.
The concept of a gene regulatory network implies that cascades of genes encoding transcription factors and their corresponding cis-regulatory elements are interlinked with each other, receive input from signaling genes, and control the expression of effector gene batteries via combinations of these cis-regulatory elements. The architecture of gene regulatory networks in development is hierarchical. They consist of kernels operating in the initial stages of development and peripheral subcircuits that control terminal cell differentiation and function. While the kernels are generally relatively stable, the peripheral subcircuits are evolutionarily more labile. Depending where a mutational change occurs in the hierarchy of a gene regulatory network the effects are more or less drastic (Erwin and Davidson, 2009).
Unfortunately, our knowledge on the molecular nature of C4-specific cis- and trans-regulatory factors is poor. We also have only a rather rudimentary understanding of which gene regulatory networks are involved in determining the development and anatomy of a typical leaf of a C3 angiosperm.
cis-Regulatory Elements
Only one cis-regulatory element for cell-specific leaf parenchyma gene expression has been described so far at the level of nucleotide resolution. The identified module consists of 41 bp and determines the mesophyll specificity of phosphoenolpyruvate carboxylase gene expression in C4 Flaveria species. The evolutionary origin of this module is known. Only small evolutionary changes were found to be required to transform its C3 ancestral form into a mesophyll expression module (Akyildiz et al., 2007), which reinforces the evolutionary easiness of C3-to-C4 transitions. In Arabidopsis and rice (Oryza sativa) quite large numbers of enhancer trap lines have been described and also screened for leaf-specific expression patterns (Springer, 2000). However, no enhancers specific for mesophyll or bundle sheath cells have been molecularly identified yet. Our knowledge about cis-regulatory elements involved in leaf parenchyma-specific gene expression remains therefore unsatisfactory.
trans-Regulatory Factors
Knowledge about trans-regulatory components operating in gene regulatory network that specify leaf anatomy or regulate parenchyma-specific gene expression is similarly fragmentary. The kernel of the gene regulatory system controlling adaxial-abaxial leaf polarity, i.e. the transcription factors of the class III homeodomain zipper and KANADI proteins, has been identified (Braybrook and Kuhlemeier, 2010). However, we have no information about the subcircuits that regulate differentiation of mesophyll and bundle sheath cells and their transcriptional networks. The GOLDEN2-LIKE (GLK) transcription factors GLK1 and GLK2 are the only exception. In Arabidopsis the GLK proteins are largely redundant and control the expression of more than 100 genes the majority of which is functionally connected with photosynthesis. In the C4 species maize, however, the two GLK genes are differentially expressed with GOLDEN2 specifically affecting only chloroplast development in the bundle sheath cells (Waters and Langdale, 2009). GLK proteins appear to have been recruited to a gene regulatory network of mesophyll/bundle sheath differentiation during maize evolution.
OUTLOOK
The world of the 21st century will face massive problems in supplying the food that is needed to nourish the growing human population and to fulfill the change in diet toward more meat consumption. Green energy from plant biomass is expected to solve at least partly our energy demands. The challenge will be to increase crop production drastically both in terms of harvestable yield and total biomass, but in a sustainable manner. C4 plants with their high photosynthetic capacity and their efficient use of nitrogen and water resources have received an increasing interest in recent years, and attempts are under way in even transferring this type of photosynthesis into current C3 crops (Sheehy et al., 2007). For embarking on this endeavor in synthetic biology we need to know the genetic architecture of C4 photosynthesis and the underlying gene regulatory networks. Understanding the evolutionary trajectories of C4 photosynthesis is a prerequisite for this exercise in synthetic experimental evolution (Erwin and Davidson, 2009).
Acknowledgments
We apologize to all those whose work could not be cited due to space constraints.
Footnotes
↵1 This work was supported by the Deutsche Forschungsgemeinschaft through the Comprehensive Research Center SFB590, the Research Group FOR1186, and the Bill and Melinda Gates Foundation.
- Received June 25, 2010.
- Accepted July 3, 2010.
- Published October 6, 2010.
LITERATURE CITED
