Explore the vast range of titles available.

C4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic and analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C2 photosynthesis) is a prerequisite for the evolution of C4. However, a mechanistic model explaining the tight connection between the evolution of C4 and C2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C3, C3–C4, and C4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires anaplerotic reactions that resemble at least parts of a basic C4 cycle. Our findings thus show how C2 photosynthesis represents a pre-adaptation for the C4 system, where the evolution of the C2 system establishes important C4 components as a side effect. Environmental pressures sometimes cause different organisms to independently evolve the same traits. A dramatic example of this phenomenon, which is called convergent evolution, can be seen in the modes used by plants to convert carbon dioxide from the air into starch during photosynthesis. Early plants existed in an environment with high levels of carbon dioxide in the air. Over time, carbon dioxide levels decreased, so plants evolved more efficient types of photosynthesis to cope. A very efficient type of photosynthesis, called C4 photosynthesis essentially represents a carbon dioxide concentration mechanism. It has evolved at least 62 times independently in 19 different families of flowering plants. Scientists have shown that a less advanced, low-efficiency version of photosynthetic carbon dioxide concentration, called C2 photosynthesis, is a stepping-stone to C4 photosynthesis. It is also known that the evolution of C4 photosynthesis required changes to the expression patterns of thousands of genes, but the exact mechanism that leads from C2 photosynthesis to C4 photosynthesis is not clear. To explore this in greater detail, Mallmann, Heckmann et al. studied plants from the genus Flaveria, which belongs to the same family as sunflowers and asters. Under identical greenhouse conditions, plants that use three different photosynthetic pathways—C3 photosynthesis, C4 photosynthesis, or an intermediate between the two—were grown and their gene expression patterns were compared. Computer simulations were used to model the metabolism of plants that relied on C2 photosynthesis. Based on the modeling, it appears that C2 photosynthesis shifts the balance of nitrogen metabolism between two types of cell that are critical to photosynthesis. To rebalance the nitrogen, several genes are expressed to trigger an ammonia recycling mechanism. The same genes are turned on during C4 photosynthesis, and this recycling mechanism include parts of the C4 process. The findings of Mallmann, Heckmann et al. suggest that the initial steps in C4 photosynthesis evolved to prevent nitrogen imbalance. Over time, this mechanism was co-opted to become part of a more efficient form of photosynthesis, which may explain why so many different plants evolved from C2 to C4 photosynthesis.

Selective pressure exerted by a massive decline in atmospheric CO₂ levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C₄ photosynthesis. C₄ species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C₄ trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C₃ and C₄ species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C₃ to C₃-C₄ intermediate to C₄ species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C₄-associated characteristics. Single gene analysis defines the C₄ cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C₄ associated pathways. Molecular evidence for a photorespiratory CO₂ pump prior to the establishment of the C₄ cycle-based CO₂ pump is provided. Cluster analysis defines the upper limit of C₄-related gene expression changes in mature leaves of Flaveria as 3582 alterations.

Pyruvate transporter is BASS2 Many of the plastid-localized metabolic pathways of plants, including the C 4 photosynthetic pathway that operates in many crop plants, depend critically on the import of pyruvate. The pyruvate transporter has proved elusive, but has now been identified as the bile acid:sodium symporter family protein 2 (BASS2). The BASS2 protein is found in the chloroplast envelope membrane, and is highly abundant in C 4 plants. Orthologues of BASS2 are present in all the genomes of land plants characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import in plastids. Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids 1 , terpenoids 2 and branched-chain amino acids 3 . In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers 4 , which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C 3 and C 4 plants of the genera Flaveria and Cleome , here we have identified a novel gene that is abundant in C 4 species, named BASS2 ( BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2 ). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C 4 plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.

Most terrestrial plants use C₃ photosynthesis to fix carbon. In multiple plant lineages a modified system known as C₄ photosynthesis has evolved. To better understand the molecular patterns associated with induction of C₄ photosynthesis, the genus Flaveria that contains C₃ and C₄ species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C₃ and two C₄ Flaveria species. Key C₄ traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C₄ photosynthesis were quantified. Candidate genes underlying each of these C₄ characteristics were identified. Principal components analysis indicated that leaf maturation and the photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over-represented for a prolonged period in the C₄ species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up-regulated in diverse C₄ species. The analysis identifies similar patterns of expression in independent C₄ lineages and so indicates that the complex C₄ pathway is associated with parallel as well as convergent evolution.

In C₄ plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C₄ dicot Flaveria bidentis grown at a low (LI; 150 μmol quanta m² s⁻¹) and medium (MI; 500 μmol quanta m² s⁻¹) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO₂ assimilation per leaf area measured at 1500 μmmol quanta m² s⁻¹ were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO₂ ({phi}) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. {phi} increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (Sm) and bundle sheath surface area per unit leaf area (Sb) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO₂ diffusion were greater in MI compared with LI plants. Despite being a C₄ species, F. bidentis is very plastic with respect to growth irradiance.

C₄ photosynthesis evolved independently numerous times, probably in response to declining atmospheric CO₂ concentrations, but also to high temperatures and aridity, which enhance water losses through transpiration. Here, the environmental factors controlling stomatal behaviour of leaf-level carbon and water exchange were examined across the evolutionary continuum from C₃ to C₄ photosynthesis at current (400 μmol mol⁻¹) and low (280 μmol mol⁻¹) atmospheric CO₂ conditions. To this aim, a stomatal optimization model was further developed to describe the evolutionary continuum from C₃ to C₄ species within a unified framework. Data on C₃, three categories of C₃–C₄ intermediates, and C₄ Flaveria species were used to parameterize the stomatal model, including parameters for the marginal water use efficiency and the efficiency of the CO₂-concentrating mechanism (or C₄ pump); these two parameters are interpreted as traits reflecting the stomatal and photosynthetic adjustments during the C₃ to C₄ transformation. Neither the marginal water use efficiency nor the C₄ pump strength changed significantly from C₃ to early C₃–C₄ intermediate stages, but both traits significantly increased between early C₃–C₄ intermediates and the C₄-like intermediates with an operational C₄ cycle. At low CO₂, net photosynthetic rates showed continuous increases from a C₃ state, across the intermediates and towards C₄ photosynthesis, but only C₄-like intermediates and C₄ species (with an operational C₄ cycle) had higher water use efficiencies than C₃ Flaveria. The results demonstrate that both the marginal water use efficiency and the C₄ pump strength increase in C₄ Flaveria to improve their photosynthesis and water use efficiency compared with C₃ species. These findings emphasize that the advantage of the early intermediate stages is predominantly carbon based, not water related.

The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO 2 pump, termed C 2 photosynthesis. How C 3 species evolved C 2 metabolism is critical to understanding the initial phases of C 4 plant evolution. To evaluate early events in C 4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C 3 and C 2 species of Flaveria, a model genus for C 4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C 3 species that are most closely related to the C 2 Flaveria species, would show rudimentary characteristics of C 2 physiology. Compared with less-related C 3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C 2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO 2 compensation point of photosynthesis was slightly lower than in the less-related C 3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO 2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO 2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C 2 photosynthesis.

C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.

The first product of sulfate assimilation in plants, cysteine, is a proteinogenic amino acid and a source of reduced sulfur for plant metabolism. Cysteine synthesis is the convergence point of the three major pathways of primary metabolism: carbon, nitrate, and sulfate assimilation. Despite the importance of metabolic and genetic coordination of these three pathways for nutrient balance in plants, the molecular mechanisms underlying this coordination, and the sensors and signals, are far from being understood. This is even more apparent in C₄ plants, where coordination of these pathways for cysteine synthesis includes the additional challenge of differential spatial localization. Here we review the coordination of sulfate, nitrate, and carbon assimilation, and show how they are altered in C₄ plants. We then summarize current knowledge of the mechanisms of coordination of these pathways. Finally, we identify urgent questions to be addressed in order to understand the integration of sulfate assimilation with carbon and nitrogen metabolism particularly in C₄ plants. We consider answering these questions to be a prerequisite for successful engineering of C₄ photosynthesis into C₃ crops to increase their efficiency.

Photorespiration raises cellular CO2 levels about 3-fold in leaves of C3–C4 intermediate Flaveria species. This was shown by using 14C-based fluxomics to determine the Rubisco in vivo carboxylation-to-oxygenation ratios.