The Calvin-Benson-Bassham cycle in C4 and Crassulacean acid metabolism species.

The Calvin-Benson-Bassham (CBB) cycle is the ancestral CO2 assimilation pathway and is found in all photosynthetic organisms. Biochemical extensions to the CBB cycle have evolved that allow the resulting pathways to act as CO2 concentrating mechanisms, either spatially in the case of C4 photosynthesis or temporally in the case of Crassulacean acid metabolism (CAM). While the biochemical steps in the C4 and CAM pathways are known, questions remain on their integration and regulation with CBB cycle activity. The application of omic and transgenic technologies is providing a more complete understanding of the biochemistry of C4 and CAM species and will also provide insight into the CBB cycle in these plants. As the global population increases, new solutions are required to increase crop yields and meet demands for food and other bioproducts. Previous work in C3 species has shown that increasing carbon assimilation through genetic manipulation of the CBB cycle can increase biomass and yield. There may also be options to improve photosynthesis in species using C4 photosynthesis and CAM through manipulation of the CBB cycle in these plants. This is an underexplored strategy and requires more basic knowledge of CBB cycle operation in these species to enable approaches for increased productivity.

Leaf transcriptomes from C3, C3-C4 intermediate, and C4 Neurachne species give insights into C4 photosynthesis evolution.

The C4 photosynthetic pathway is hypothesized to have evolved from the ancestral C3 pathway through progressive changes in leaf anatomy and biochemistry with extant C3-C4 photosynthetic intermediate species representing phenotypes between species demonstrating full C3 and full C4 states. The Australian endemic genus Neurachne is the only known grass group that contains distinct, closely related species that carry out C3, C3-C4 intermediate, or C4 photosynthesis. To explore and understand the molecular mechanisms underlying C4 photosynthesis evolution in this genus, leaf transcriptomes were generated from two C3, three photosynthetic intermediate (proto-Kranz, C2-like, and C2), and two C4  Neurachne species. The data were used to reconstruct phylogenetic relationships in Neurachne, which confirmed two independent C4 origins in the genus. Relative transcript abundances substantiated the photosynthetic phenotypes of individual species and highlighted transcriptional investment differences between species, including between the two C4 species. The data also revealed proteins potentially involved in C4 cycle intermediate transport and identified molecular mechanisms responsible for the evolution of C4-associated proteins in the genus.

Tribulus (Zygophyllaceae) as a case study for the evolution of C2 and C4 photosynthesis.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

Metabolic profiles in C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3-C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae.

The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C * values of 56-61 µmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C * of 44 µmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.

C4-like photosynthesis and the effects of leaf senescence on C4-like physiology in Sesuvium sesuvioides (Aizoaceae).

Sesuvium sesuvioides (Sesuvioideae, Aizoaceae) is a perennial, salt-tolerant herb distributed in flats, depressions, or disturbed habitats of southern Africa and the Cape Verdes. Based on carbon isotope values, it is considered a C4 species, despite a relatively high ratio of mesophyll to bundle sheath cells (2.7:1) in the portulacelloid leaf anatomy. Using leaf anatomy, immunocytochemistry, gas exchange measurements, and enzyme activity assays, we sought to identify the biochemical subtype of C4 photosynthesis used by S. sesuvioides and to explore the anatomical, physiological, and biochemical traits of young, mature, and senescing leaves, with the aim to elucidate the plasticity and possible limitations of the photosynthetic efficiency in this species. Assays indicated that S. sesuvioides employs the NADP-malic enzyme as the major decarboxylating enzyme. The activity of C4 enzymes, however, declined as leaves aged, and the proportion of water storage tissue increased while air space decreased. These changes suggest a functional shift from photosynthesis to water storage in older leaves. Interestingly, S. sesuvioides demonstrated CO2 compensation points ranging between C4 and C3-C4 intermediate values, and immunocytochemistry revealed labeling of the Rubisco large subunit in mesophyll cells. We hypothesize that S. sesuvioides represents a young C4 lineage with C4-like photosynthesis in which C3 and C4 cycles are running simultaneously in the mesophyll.

Metabolite profiles reveal interspecific variation in operation of the Calvin-Benson cycle in both C4 and C3 plants.

Low atmospheric CO2 in recent geological time led to the evolution of carbon-concentrating mechanisms (CCMs) such as C4 photosynthesis in >65 terrestrial plant lineages. We know little about the impact of low CO2 on the Calvin-Benson cycle (CBC) in C3 species that did not evolve CCMs, representing >90% of terrestrial plant species. Metabolite profiling provides a top-down strategy to investigate the operational balance in a pathway. We profiled CBC intermediates in a panel of C4 (Zea mays, Setaria viridis, Flaveria bidentis, and F. trinervia) and C3 species (Oryza sativa, Triticium aestivum, Arabidopsis thaliana, Nicotiana tabacum, and Manihot esculenta). Principal component analysis revealed differences between C4 and C3 species that were driven by many metabolites, including lower ribulose 1,5-bisphosphate in C4 species. Strikingly, there was also considerable variation between C3 species. This was partly due to different chlorophyll and protein contents, but mainly to differences in relative levels of metabolites. Correlation analysis indicated that one contributory factor was the balance between fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and Rubisco. Our results point to the CBC having experienced different evolutionary trajectories in C3 species since the ancestors of modern plant lineages diverged. They underline the need to understand CBC operation in a wide range of species.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne.

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct ß-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all ß-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.

C3 cotyledons are followed by C4 leaves: intra-individual transcriptome analysis of Salsola soda (Chenopodiaceae).

Some species of Salsoleae (Chenopodiaceae) convert from C3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.

C3-C4 intermediacy in grasses: organelle enrichment and distribution, glycine decarboxylase expression, and the rise of C2 photosynthesis.

Photorespiratory glycine shuttling and decarboxylation in bundle sheath (BS) cells exhibited by C2 species is proposed to be the evolutionary bridge to C4 photosynthesis in eudicots. To evaluate this in grasses, we compare anatomy, cellular localization of glycine decarboxylase (GDC), and photosynthetic physiology of a suspected C2 grass, Homolepis aturensis, with these traits in known C2 grasses, Neurachne minor and Steinchisma hians, and C3 S laxum that is sister to S hians We also use publicly available genome and RNA-sequencing data to examine the evolution of GDC subunits and enhance our understanding of the evolution of BS-specific GDC expression in C2 and C4 grasses. Our results confirm the identity of H aturensis as a C2 species; GDC is confined predominantly to the organelle-enriched BS cells in H aturensis and S hians and to mestome sheath cells of N minor Phylogenetic analyses and data obtained from immunodetection of the P-subunit of GDC are consistent with the hypothesis that the BS dominant levels of GDC in C2 and C4 species are due to changes in expression of a single GLDP gene in M and BS cells. All BS mitochondria and peroxisomes and most chloroplasts in H aturensis and S hians are situated centripetally in a pattern identical to C2 eudicots. In S laxum, which has C3-like gas exchange patterns, mitochondria and peroxisomes are positioned centripetally as they are in S hians This subcellular phenotype, also present in eudicots, is posited to initiate a facilitation cascade leading to C2 and C4 photosynthesis.

High-resolution secondary ion mass spectrometry analysis of carbon dynamics in mycorrhizas formed by an obligately myco-heterotrophic orchid.

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants.

The evolution of C(4) photosynthesis from C(3) ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C(3), C(3) -C(4) intermediate and C(4) species from 12 lineages of C(4) evolution. All C(3) species had more chloroplasts per M cell area than their C(4) relatives in high-light growth conditions. C(3) species also had higher chloroplast coverage of the M cell periphery than C(4) species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C(4) lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C(4) species than their C(3) relatives. C(3) -C(4) intermediate species generally exhibited similar patterns as their C(3) relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C(4) function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C(4) M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C(4) photosynthesis.

Biochemical characterization of the chloroplastic ß-carbonic anhydrase from Flaveria bidentis (L.) "Kuntze".

C3 and C4 plant carbonic anhydrases (CAs) are zinc-enzymes that catalyze the reversible hydration of CO2. They are sub-divided in three classes: α, ß and γ, being distributed between both photosynthetic subtypes. The C4 dicotyledon species Flaveria bidentis (L.) "Kuntze" contains a small gene family encoding three distinct ß-CAs, named FbiCA1, FbiCA2 and FbiCA3. We have expressed and purified recombinant FbiCA1, which is localized in the chloroplast where it is thought to play a role in lipid biosynthesis and antioxidant activity, and biochemically characterized it by spectroscopic and inhibition experiments. FbiCA1 is a compact octameric protein that is moderately inhibited by carboxylate molecules. Surprisingly, pyruvate, but not lactate, did not inhibit FbiCA1 at concentrations up to 10 mM, suggesting that its capacity to tolerate high pyruvate concentration reflects the high concentration of pyruvate in the chloroplasts of bundle-sheath and mesophyll cells involved in C4 photosynthesis.

Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels.

The biochemistry and leaf anatomy of plants using C4 photosynthesis promote the concentration of atmospheric CO2 in leaf tissue that leads to improvements in growth and yield of C4 plants over C3 species in hot, dry, high light, and/or saline environments. C4 plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C4 photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C3 photosynthesis. In addition to C3 and C4 species, some plant lineages contain closely related C3-C4 intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C3 plants and species using C4 photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C4 photosynthesis. It is now clear that great variation exists in C4 leaf anatomy, and diverse molecular mechanisms underlie C4 biochemistry and physiology. However, all these different paths have led to the same destination-the expression of a C4 CO2 concentrating mechanism. Further identification of C4 leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.

Shoot atmospheric contact is of little importance to aeration of deeper portions of the wetland plant Meionectes brownii; submerged organs mainly acquire O2 from the water column or produce it endogenously in underwater photosynthesis.

Partial shoot submergence is considered less stressful than complete submergence of plants, as aerial contact allows gas exchange with the atmosphere. In situ microelectrode studies of the wetland plant Meionectes brownii showed that O(2) dynamics in the submerged stems and aquatic roots of partially submerged plants were similar to those of completely submerged plants, with internal O(2) concentrations in both organs dropping to less than 5 kPa by dawn regardless of submergence level. The anatomy at the nodes and the relationship between tissue porosity and rates of O(2) diffusion through stems were studied. Stem internodes contained aerenchyma and had mean gas space area of 17.7% per cross section, whereas nodes had 8.2%, but nodal porosity was highly variable, some nodes had very low porosity or were completely occluded (ca. 23% of nodes sampled). The cumulative effect of these low porosity nodes would have impeded internal O(2) movement down stems. Therefore, regardless of the presence of an aerial connection, the deeper portions of submerged organs sourced most of their O(2) via inwards diffusion from the water column during the night, and endogenous production in underwater photosynthesis during the daytime.

Kinetic and anion inhibition studies of a ß-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis.

Several ß-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C4 plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO2 hydrase catalytic activity (kcat of 1.2×10(5) and kcat/Km of 7.5×10(6)M(-1)×s(-1)) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (KIs in the range of 4-60µM). Such inhibitors may be used as tools to better understand the role of various ß-CA isoforms in photosynthesis.

Carbonic anhydrase and the molecular evolution of C4 photosynthesis.

C(4) photosynthesis, a biochemical CO(2)-concentrating mechanism (CCM), evolved more than 60 times within the angiosperms from C(3) ancestors. The genus Flaveria, which contains species demonstrating C(3), C(3)-C(4), C(4)-like or C(4) photosynthesis, is a model for examining the molecular evolution of the C(4) pathway. Work with carbonic anhydrase (CA), and C(3) and C(4) Flaveria congeners has added significantly to the understanding of this process. The C(4) form of CA3, a ß-CA, which catalyses the first reaction in the C(4) pathway by hydrating atmospheric CO(2) to bicarbonate in the cytosol of mesophyll cells (mcs), evolved from a chloroplastic C(3) ancestor. The molecular modifications to the ancestral CA3 gene included the loss of the sequence encoding the chloroplast transit peptide, and mutations in regulatory regions that resulted in high levels of expression in the C(4) mesophyll. Analyses of the CA3 proteins and regulatory elements from Flaveria photosynthetic intermediates indicated C(4) biochemistry very likely evolved in a specific, stepwise manner in this genus. The details of the mechanisms involved in the molecular evolution of other C(4) plant ß-CAs are unknown; however, comparative genetics indicate gene duplication and neofunctionalization played significant roles as they did in Flaveria.

Multiple photosynthetic transitions, polyploidy, and lateral gene transfer in the grass subtribe Neurachninae.

The Neurachninae is the only grass lineage known to contain C(3), C(4), and C(3)-C(4) intermediate species, and as such has been suggested as a model system for studies of photosynthetic pathway evolution in the Poaceae; however, a lack of a robust phylogenetic framework has hindered this possibility. In this study, plastid and nuclear markers were used to reconstruct evolutionary relationships among Neurachninae species. In addition, photosynthetic types were determined with carbon isotope ratios, and genome sizes with flow cytometry. A high frequency of autopolyploidy was found in the Neurachninae, including in Neurachne munroi F.Muell. and Paraneurachne muelleri S.T.Blake, which independently evolved C(4) photosynthesis. Phylogenetic analyses also showed that following their separate C(4) origins, these two taxa exchanged a gene encoding the C(4) form of phosphoenolpyruvate carboxylase. The C(3)-C(4) intermediate Neurachne minor S.T.Blake is phylogenetically distinct from the two C(4) lineages, indicating that intermediacy in this species evolved separately from transitional stages preceding C(4) origins. The Neurachninae shows a substantial capacity to evolve new photosynthetic pathways repeatedly. Enablers of these transitions might include anatomical pre-conditions in the C(3) ancestor, and frequent autopolyploidization. Transfer of key C(4) genetic elements between independently evolved C(4) taxa may have also facilitated a rapid adaptation of photosynthesis in these grasses that had to survive in the harsh climate appearing during the late Pliocene in Australia.