The operation of PEPCK increases light harvesting plasticity in C4 NAD-ME and NADP-ME photosynthetic subtypes: A theoretical study.

The repeated emergence of NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvate carboxykinase (PEPCK) subtypes of C4 photosynthesis are iconic examples of convergent evolution, which suggests that these biochemistries do not randomly assemble, but are instead specific adaptations resulting from unknown evolutionary drivers. Theoretical studies that are based on the classic biochemical understanding have repeatedly proposed light-use efficiency as a possible benefit of the PEPCK subtype. However, quantum yield measurements do not support this idea. We explore this inconsistency here via an analytical model that features explicit descriptions across a seamless gradient between C4 biochemistries to analyse light harvesting and dark photosynthetic metabolism. Our simulations show that the NADP-ME subtype, operated by the most productive crops, is the most efficient. The NAD-ME subtype has lower efficiency, but has greater light harvesting plasticity (the capacity to assimilate CO2 in the broadest combination of light intensity and spectral qualities). In both NADP-ME and NAD-ME backgrounds, increasing PEPCK activity corresponds to greater light harvesting plasticity but likely imposed a reduction in photosynthetic efficiency. We draw the first mechanistic links between light harvesting and C4 subtypes, providing the theoretical basis for future investigation.

Leaf anatomy explains the strength of C4 activity within the grass species Alloteropsis semialata.

C4 photosynthesis results from anatomical and biochemical characteristics that together concentrate CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), increasing productivity in warm conditions. This complex trait evolved through the gradual accumulation of components, and particular species possess only some of these, resulting in weak C4 activity. The consequences of adding C4 components have been modelled and investigated through comparative approaches, but the intraspecific dynamics responsible for strengthening the C4 pathway remain largely unexplored. Here, we evaluate the link between anatomical variation and C4 activity, focusing on populations of the photosynthetically diverse grass Alloteropsis semialata that fix various proportions of carbon via the C4 cycle. The carbon isotope ratios in these populations range from values typical of C3 to those typical of C4 plants. This variation is statistically explained by a combination of leaf anatomical traits linked to the preponderance of bundle sheath tissue. We hypothesize that increased investment in bundle sheath boosts the strength of the intercellular C4 pump and shifts the balance of carbon acquisition towards the C4 cycle. Carbon isotope ratios indicating a stronger C4 pathway are associated with warmer, drier environments, suggesting that incremental anatomical alterations can lead to the emergence of C4 physiology during local adaptation within metapopulations.

Evolutionary implications of C2 photosynthesis: how complex biochemical trade-offs may limit C4 evolution.

The C2 carbon-concentrating mechanism increases net CO2 assimilation by shuttling photorespiratory CO2 in the form of glycine from mesophyll to bundle sheath cells, where CO2 concentrates and can be re-assimilated. This glycine shuttle also releases NH3 and serine into the bundle sheath, and modelling studies suggest that this influx of NH3 may cause a nitrogen imbalance between the two cell types that selects for the C4 carbon-concentrating mechanism. Here we provide an alternative hypothesis outlining mechanisms by which bundle sheath NH3 and serine play vital roles to not only influence the status of C2 plants along the C3 to C4 evolutionary trajectory, but to also convey stress tolerance to these unique plants. Our hypothesis explains how an optimized bundle sheath nitrogen hub interacts with sulfur and carbon metabolism to mitigate the effects of high photorespiratory conditions. While C2 photosynthesis is typically cited for its intermediary role in C4 photosynthesis evolution, our alternative hypothesis provides a mechanism to explain why some C2 lineages have not made this transition. We propose that stress resilience, coupled with open flux tricarboxylic acid and photorespiration pathways, conveys an advantage to C2 plants in fluctuating environments.

C4 trees have a broader niche than their close C3 relatives.

Previous studies have demonstrated the ecological sorting of herbaceous C3 and C4 species along gradients of precipitation and temperature: C4 herbaceous species typically occupy drier and warmer environments than their C3 relatives. However, it is unclear if this pattern holds true for C4 tree species, which are unique to Euphorbiaceae and found only on the Hawaiian Islands. Here, we combine occurrence data with local environmental and soil datasets to, for the first time, distinguish the ecological factors associated with photosynthetic diversification in the tree life form. These data are presented within a phylogenetic framework. We show that C3 and C4 trees inhabit similar environments, but that C4 photosynthesis expands the ecological niche in trees relative to that of C3 tree species. In particular, when compared with C3 trees, C4 trees moved into higher elevation habitats with characteristically sparse vegetation (and thus greater sunlight) and cooler temperatures, a pattern which contrasts with that of herbaceous species. Understanding the relationship between C4 photosynthesis and ecological niche in tree species has implications for establishing how C4 photosynthesis has, in this rare instance, evolved in trees, and whether this unique combination of traits could be exploited from an engineering perspective.

Cellular perspectives for improving mesophyll conductance.

After entering the leaf, CO2 faces an intricate pathway to the site of photosynthetic fixation embedded within the chloroplasts. The efficiency of CO2 flux is hindered by a number of structural and biochemical barriers which, together, define the ease of flow of the gas within the leaf, termed mesophyll conductance. Previous authors have identified the key elements of this pathway, raising the prospect of engineering the system to improve CO2 flux and, thus, to increase leaf photosynthetic efficiency. In this review, we provide a perspective on the potential for improving the individual elements that contribute to this complex parameter. We lay particular emphasis on generation of the cellular architecture of the leaf which sets the initial boundaries of a number of mesophyll conductance parameters, incorporating an overview of the molecular transport processes which have been proposed as major facilitators of CO2 flux across structural boundaries along the pathway. The review highlights the research areas where future effort might be invested to increase our fundamental understanding of mesophyll conductance and leaf function and, consequently, to enable translation of these findings to improve the efficiency of crop photosynthesis.

Low dispersal and ploidy differences in a grass maintain photosynthetic diversity despite gene flow and habitat overlap.

Geographical isolation facilitates the emergence of distinct phenotypes within a single species, but reproductive barriers or selection are needed to maintain the polymorphism after secondary contact. Here, we explore the processes that maintain intraspecific variation of C4 photosynthesis, a complex trait that results from the combined action of multiple genes. The grass Alloteropsis semialata includes C4 and non-C4 populations, which have coexisted as a polyploid series for more than 1 million years in the miombo woodlands of Africa. Using population genomics, we show that there is genome-wide divergence for the photosynthetic types, but the current geographical distribution does not reflect a simple habitat displacement scenario as the genetic clusters overlap, being occasionally mixed within a given habitat. Despite evidence of recurrent introgression between non-C4 and C4 groups, in both diploids and polyploids, the distinct genetic lineages retain their identity, potentially because of selection against hybrids. Coupled with strong isolation by distance within each genetic group, this selection created a geographical mosaic of photosynthetic types. Diploid C4 and non-C4 types never grew together, and the C4 type from mixed populations constantly belonged to the hexaploid lineage. By limiting reproductive interactions between photosynthetic types, the ploidy difference probably allows their co-occurrence, reinforcing the functional diversity within this species. Together, these factors enabled the persistence of divergent physiological traits of ecological importance within a single species despite gene flow and habitat overlap.

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.

C2 photosynthesis: a promising route towards crop improvement?

C2 photosynthesis is a carbon concentrating mechanism that can increase net CO2 assimilation by capturing, concentrating and re-assimilating CO2 released by photorespiration. Empirical and modelling studies indicate that C2 plants assimilate more carbon than C3 plants under high temperature, bright light, and low CO2 conditions. I argue that engineering C2 photosynthesis into C3 crops is a promising approach to improve photosynthetic performance under these - and temporally heterogeneous - environments, and review the modifications that may re-create a C2 phenotype in C3 plants. Although a C2 engineering program would encounter many of the same challenges faced by C4 engineering programmes, the simpler leaf anatomical requirements make C2 engineering a feasible approach to improve crops in the medium term.

Why is C4 photosynthesis so rare in trees?

Since C4 photosynthesis was first discovered >50 years ago, researchers have sought to understand how this complex trait evolved from the ancestral C3 photosynthetic machinery on >60 occasions. Despite its repeated emergence across the plant kingdom, C4 photosynthesis is notably rare in trees, with true C4 trees only existing in Euphorbia. Here we consider aspects of the C4 trait that could limit but not preclude the evolution of a C4 tree, including reduced quantum yield, increased energetic demand, reduced adaptive plasticity, evolutionary constraints, and a new theory that the passive symplastic phloem loading mechanism observed in trees, combined with difficulties in maintaining sugar and water transport over a long pathlength, could make C4 photosynthesis largely incompatible with the tree lifeform. We conclude that the transition to a tree habit within C4 lineages as well as the emergence of C4 photosynthesis within pre-existing trees would both face a series of challenges that together explain the global rarity of C4 photosynthesis in trees. The C4 trees in Euphorbia are therefore exceptional in how they have circumvented every potential barrier to the rare C4 tree lifeform.

C4 anatomy can evolve via a single developmental change.

C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.

Bundle sheath chloroplast volume can house sufficient Rubisco to avoid limiting C4 photosynthesis during chilling.

C4 leaves confine Rubisco to bundle sheath cells. Thus, the size of bundle sheath compartments and the total volume of chloroplasts within them limit the space available for Rubisco. Rubisco activity limits photosynthesis at low temperatures. C3 plants counter this limitation by increasing leaf Rubisco content, yet few C4 species do the same. Because C3 plants usually outperform C4 plants in chilling environments, it has been suggested that there is insufficient chloroplast volume available in the bundle sheath of C4 leaves to allow such an increase in Rubisco at low temperatures. We investigated this potential limitation by measuring bundle sheath and mesophyll compartment volumes and chloroplast contents, as well as leaf thickness and inter-veinal distance, in three C4 Andropogoneae grasses: two crops (Zea mays and Saccharum officinarum) and a wild, chilling-tolerant grass (Miscanthus × giganteus). A wild C4 Paniceae grass (Alloteropsis semialata) was also included. Despite significant structural differences between species, there was no evidence of increased bundle sheath chloroplast volume per leaf area available to the chilling-tolerant species, relative to the chilling-sensitive ones. Maximal theoretical photosynthetic capacity of the leaf far exceeded the photosynthetic rates achieved even at low temperatures. C4 bundle sheath cells therefore have the chloroplast volume to house sufficient Rubisco to avoid limiting C4 photosynthesis during chilling.

Key changes in gene expression identified for different stages of C4 evolution in Alloteropsis semialata.

C4 photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared with C3 species, the C4 state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically informed comparative transcriptomics show that intermediates with a weak C4 cycle are separated from the C3 phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C4 enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxylase. The subsequent transition to full C4 physiology was accompanied by increases in another 15 genes (0.06%), including only the core C4 enzyme pyruvate orthophosphate dikinase. These changes probably created a rudimentary C4 physiology, and isolated populations subsequently improved this emerging C4 physiology, resulting in a patchwork of expression for some C4 accessory genes. Our work shows how C4 assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous step. These create short bridges across adaptive landscapes that probably facilitated the recurrent origins of C4 photosynthesis through a gradual process of evolution.

Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity.

The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.

Despite phylogenetic effects, C3-C4 lineages bridge the ecological gap to C4 photosynthesis.

C4 photosynthesis is a physiological innovation involving several anatomical and biochemical components that emerged recurrently in flowering plants. This complex trait evolved via a series of physiological intermediates, broadly termed 'C3-C4', which have been widely studied to understand C4 origins. While this research program has focused on biochemistry, physiology, and anatomy, the ecology of these intermediates remains largely unexplored. Here, we use global occurrence data and local habitat descriptions to characterize the niches of multiple C3-C4 lineages, as well as their close C3 and C4 relatives. While C3-C4 taxa tend to occur in warm climates, their abiotic niches are spread along other dimensions, making it impossible to define a universal C3-C4 niche. Phylogeny-based comparisons suggest that, despite shifts associated with photosynthetic types, the precipitation component of the C3-C4 niche is particularly lineage specific, being highly correlated with that of closely related C3 and C4 taxa. Our large-scale analyses suggest that C3-C4 lineages converged toward warm habitats, which may have facilitated the transition to C4 photosynthesis, effectively bridging the ecological gap between C3 and C4 plants. The intermediates retained some precipitation aspects of their C3 ancestors' habitat, and likely transmitted them to their C4 descendants, contributing to the diversity among C4 lineages seen today.

Anatomical constraints to C4 evolution: light harvesting capacity in the bundle sheath.

In C4 photosynthesis CO2 assimilation and reduction are typically coordinated across mesophyll (M) and bundle sheath (BS) cells, respectively. This system consequently requires sufficient light to reach BS to generate enough ATP to allow ribulose-1,5-bisphosphate (RuBP) regeneration in BS. Leaf anatomy influences BS light penetration and therefore constrains C4 cycle functionality. Using an absorption scattering model (coded in Excel, and freely downloadable) we simulate light penetration profiles and rates of ATP production in BS across the C3 , C3 -C4 and C4 anatomical continua. We present a trade-off for light absorption between BS pigment concentration and space allocation. C3 BS anatomy limits light absorption and benefits little from high pigment concentrations. Unpigmented BS extensions increase BS light penetration. C4 and C3 -C4 anatomies have the potential to generate sufficient ATP in the BS, whereas typical C3 anatomy does not, except some C3 taxa closely related to C4 groups. Insufficient volume of BS, relative to M, will hamper a C4 cycle via insufficient BS light absorption. Thus, BS ATP production and RuBP regeneration, coupled with increased BS investments, allow greater operational plasticity. We propose that larger BS in C3 lineages may be co-opted for C3 -C4 and C4 biochemistry requirements.

Genome biogeography reveals the intraspecific spread of adaptive mutations for a complex trait.

Physiological novelties are often studied at macro-evolutionary scales such that their micro-evolutionary origins remain poorly understood. Here, we test the hypothesis that key components of a complex trait can evolve in isolation and later be combined by gene flow. We use C4 photosynthesis as a study system, a derived physiology that increases plant productivity in warm, dry conditions. The grass Alloteropsis semialata includes C4 and non-C4 genotypes, with some populations using laterally acquired C4 -adaptive loci, providing an outstanding system to track the spread of novel adaptive mutations. Using genome data from C4 and non-C4 A. semialata individuals spanning the species' range, we infer and date past migrations of different parts of the genome. Our results show that photosynthetic types initially diverged in isolated populations, where key C4 components were acquired. However, rare but recurrent subsequent gene flow allowed the spread of adaptive loci across genetic pools. Indeed, laterally acquired genes for key C4 functions were rapidly passed between populations with otherwise distinct genomic backgrounds. Thus, our intraspecific study of C4 -related genomic variation indicates that components of adaptive traits can evolve separately and later be combined through secondary gene flow, leading to the assembly and optimization of evolutionary innovations.

Evolutionary implications of C3 -C4 intermediates in the grass Alloteropsis semialata.

C4 photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C3 pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C4 -like components. Determining the adaptive value of these components is key to understanding how C4 photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C3 and C4 genotypes. Analyses of δ(13) C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C3 -C4 intermediate individuals in the species. Based on previous phylogenetic analyses, the C3 -C4 individuals are not hybrids between the C3 and C4 genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C4 descendants. C3 A. semialata, present in colder climates, likely represents a reversal from a C3 -C4 intermediate state, indicating that, unlike C4 photosynthesis, evolution of the C3 -C4 phenotype is not irreversible.

Photosynthetic innovation broadens the niche within a single species.

Adaptation to changing environments often requires novel traits, but how such traits directly affect the ecological niche remains poorly understood. Multiple plant lineages have evolved C4 photosynthesis, a combination of anatomical and biochemical novelties predicted to increase productivity in warm and arid conditions. Here, we infer the dispersal history across geographical and environmental space in the only known species with both C4 and non-C4 genotypes, the grass Alloteropsis semialata. While non-C4 individuals remained confined to a limited geographic area and restricted ecological conditions, C4 individuals dispersed across three continents and into an expanded range of environments, encompassing the ancestral one. This first intraspecific investigation of C4 evolutionary ecology shows that, in otherwise similar plants, C4 photosynthesis does not shift the ecological niche, but broadens it, allowing dispersal into diverse conditions and over long distances. Over macroevolutionary timescales, this immediate effect can be blurred by subsequent specialisation towards more extreme niches.

Deconstructing Kranz anatomy to understand C4 evolution.

C4 photosynthesis is a complex physiological adaptation that confers greater productivity than the ancestral C3 photosynthetic type in environments where photorespiration is high. It evolved in multiple lineages through the coordination of anatomical and biochemical components, which concentrate CO2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most C4 plants, the CO2-concentrating mechanism is achieved via the confinement of Rubisco to bundle-sheath cells, into which CO2 is biochemically pumped from surrounding mesophyll cells. The C4 biochemical pathway relies on a specific suite of leaf functional properties, often referred to as Kranz anatomy. These include the existence of discrete compartments differentially connected to the atmosphere, a close contact between these compartments, and a relatively large compartment to host the Calvin cycle. In this review, we use a quantitative dataset for grasses (Poaceae) and examples from other groups to isolate the changes in anatomical characteristics that generate these functional properties, including changes in the size, number, and distribution of different cell types. These underlying anatomical characteristics vary among C4 origins, as similar functions emerged via different modifications of anatomical characteristics. In addition, the quantitative characteristics of leaves all vary continuously across C3 and C4 taxa, resulting in C4-like values in some C3 taxa. These observations suggest that the evolution of C4-suitable anatomy might require relatively few changes in plant lineages with anatomical predispositions. Furthermore, the distribution of anatomical traits across C3 and C4 taxa has important implications for the functional diversity observed among C4 lineages and for the approaches used to identify genetic determinants of C4 anatomy.

Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.